ABSTRACT: Flavin cofactors are attractive Electron Spin Resonance (ESR) probes for proteins because cellular reductants and light can generate their semiquinone states. We have used ESR spectroscopy to study the bacterial transmembrane aerotaxis receptor (Aer) in its native Escherichia coli membrane environment. Optimization of the spectroscopic (electronic relaxation times) and cell growth (isotopic labeling) conditions allowed for measurements of Aer with its partners - the histidine kinase (CheA) and the coupling protein (CheW) - in native signaling arrays. Continuous-wave ESR measurements at room temperature showed a rigid Aer flavin immobilized in the cofactor pocket and Q-band electron nuclear double resonance (ENDOR) measurements identified a predominant anionic semiquinone radical state in cell. Q-band four-pulse double electron-electron resonance (4P-DEER) measurements indicated a 4.1 nm distance between the two flavins of an Aer homodimer, consistent with previous in vitro measurements, but also revealed additional separations in cell indicative of chemoreceptor arrays, not previously observed for Aer. For general application, we further developed a genetically encoded Light-Oxygen and Voltage (LOV) domain for incorporation into target proteins as an ESR probe of structural properties in cell. This approach provides a framework to elucidate protein oligomeric states and conformations that are difficult to reproduce in vitro.
Woven covalent organic frameworks (COFs) offer immense potential as photoactive materials because Cu(I) complexes are periodically integrated into the COF structure via a single synthetic step. A photoactive interlocking (woven) COF featuring Cu(I) photosensitizers that are spatially isolated and periodically arranged in three dimensions has been successfully synthesized and characterized. The optoelectronic properties of this COF, such as light absorption and photocatalytic performance toward the degradation of sulfamethoxazole (SMX) under visible light, are investigated. The reusability and stability of this COF are compared with the Cu(PDB)2BF4 complex which displayed rapid deactivation and is not reusable. Conversely, The metallo-COF is stable over several catalytic cycles, highlighting a distinct advantage of the stabilizing effects of the COF over discrete molecules.
ABSTRACT: Circadian clocks are composed of transcription-translation negative feedback loops that pace rhythms of gene expression to the diurnal cycle. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH), and Casein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the white-collar complex (WCC). FRQ orchestrates key molecular interactions of the clock despite containing little predicted tertiary structure. Spin labeling and pulse-dipolar electron spin resonance spectroscopy provide domain-specific structural insights into the 989-residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state, but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid–liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic clocks.
ABSTRACT: Woven covalent organic frameworks (COF) possess entangled 3D frameworks. The metallated version of these structures contains spatially isolated Cu(I) centers and promising optoelectronic properties because of metal-to-ligand charge transfer (MLCT). However, despite their potential, woven COFs have not yet been investigated as photocatalysts. In this study, a new woven COF, Cu-PhenBDA-COF, functionalized with diacetylene bonds is developed. Cu-PhenBDA-COF is fully characterized, and the optoelectronic and photocatalytic properties are compared to previously reported Cu-COF-505. The diacetylene bonds of the linker positively impact the optoelectronic properties of Cu-PhenBDA-COF and result in a narrower bandgap and better charge separation efficiency. When the Cu(I) center is removed from both woven COFs, the absorption edge is blueshifted, resulting in a wider bandgap, and there is a considerable decrease in the charge separation efficiency, underscoring the pivotal role of MLCT. This trend is reflected in the photocatalytic activity of the woven COFs toward the degradation of sulfamethoxazole in water, where the highest reaction rate constant (kapp) is recorded for the metallated diacetylene functionalized woven COF, Cu-PhenBDA-COF.
ABSTRACT: Woven covalent organic frameworks (COF) possess entangled 3D frameworks. The metallated version of these structures contains spatially isolated Cu(I) centers and promising optoelectronic properties because of metal-to-ligand charge transfer (MLCT). However, despite their potential, woven COFs have not yet been investigated as photocatalysts. In this study, a new woven COF, Cu-PhenBDA-COF, functionalized with diacetylene bonds is developed. Cu-PhenBDA-COF is fully characterized, and the optoelectronic and photocatalytic properties are compared to previously reported Cu-COF-505. The diacetylene bonds of the linker positively impact the optoelectronic properties of Cu-PhenBDA-COF and result in a narrower bandgap and better charge separation efficiency. When the Cu(I) center is removed from both woven COFs, the absorption edge is blueshifted, resulting in a wider bandgap, and there is a considerable decrease in the charge separation efficiency, underscoring the pivotal role of MLCT. This trend is reflected in the photocatalytic activity of the woven COFs toward the degradation of sulfamethoxazole in water, where the highest reaction rate constant (kapp) is recorded for the metallated diacetylene functionalized woven COF, Cu-PhenBDA-COF.
ABSTRACT: Electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for investigating the structure and dynamics of proteins. The introduction of paramagnetic moieties at specific positions in a protein enables precise measurement of local structure and dynamics. This technique, termed site-directed spin-labeling, has traditionally been performed using cysteine-reactive radical-containing probes. However, large proteins are more likely to contain multiple cysteine residues and cysteine labeling at specific sites may be infeasible or impede function. To address this concern, we applied three peptide-ligating enzymes (sortase, asparaginyl endopeptidase, and inteins) for nitroxide labeling of N- and C-termini of select monomeric and dimeric proteins. Continuous wave and pulsed EPR (double electron electron resonance) experiments reveal specific attachment of nitroxide probes to ether N-termini (OaAEP1) or C-termini (sortase and intein) across three test proteins (CheY, CheA, and iLOV), thereby enabling a straightforward, highly specific, and general method for protein labeling. Importantly, the linker length (3, 5, and 9 residues for OaAEP1, intein, and sortase reactions, respectively) between the probe and the target protein has a large impact on the utility of distance measurements by pulsed EPR, with longer linkers leading to broader distributions. As these methods are only dependent on accessible N- and C-termini, we anticipate application to a wide range of protein targets for biomolecular EPR spectroscopy.
ABSTRACT: Lipid droplets (LDs) are dynamic organelles that contain an oil core mainly composed of triglycerides (TAG) that is surrounded by a phospholipid monolayer and LD-associated proteins called perilipins (PLINs). During LD biogenesis, perilipin 3 (PLIN3) is recruited to nascent LDs as they emerge from the endoplasmic reticulum. Here, we analyze how lipid composition affects PLIN3 recruitment to membrane bilayers and LDs, and the structural changes that occur upon membrane binding. We find that the TAG precursors phosphatidic acid and diacylglycerol (DAG) recruit PLIN3 to membrane bilayers and define an expanded Perilipin-ADRP-Tip47 (PAT) domain that preferentially binds DAG-enriched membranes. Membrane binding induces a disorder to order transition of alpha helices within the PAT domain and 11-mer repeats, with intramolecular distance measurements consistent with the expanded PAT domain adopting a folded but dynamic structure upon membrane binding. In cells, PLIN3 is recruited to DAG-enriched ER membranes, and this requires both the PAT domain and 11-mer repeats. This provides molecular details of PLIN3 recruitment to nascent LDs and identifies a function of the PAT domain of PLIN3 in DAG binding.
ABSTRACT: Thaumatin, a potent sweet tasting protein extracted from the Katemfe Plant, is emerging as a natural alternative to synthetic non-nutritive sweeteners and flavor enhancer. As a food additive, its stability within the food matrix during thermal processing is of great interest to the food industry. When heated under neutral or basic conditions, thaumatin was found to lose its sweetness due to protein aggregation caused by sulfhydryl catalyzed disulfide bond interchange. At lower pH, while thaumatin was also found to lose sweetness after heating, it does so at a slower rate and shows more resistance to sweetness loss. SDS-PAGE indicated that thaumatin fragmented into multiple smaller pieces under heating in acidic pH. Using BEMPO-3, a lipophilic spin trap, we were able to detect the presence of a free-radical within the hydrophobic region of the protein during heating. Protein carbonyl content, a byproduct of protein oxidation, also increased upon heating, providing additional evidence for protein cleavage by a radical pathway. Hexyl gallate successfully inhibited the radical generation as well as protein carbonyl formation of thaumatin during heating.
ABSTRACT: Complexins play a critical role in regulating SNARE-mediated exocytosis of synaptic vesicles. Evolutionary divergences in complexin function have complicated our understanding of the role these proteins play in inhibiting the spontaneous fusion of vesicles. Previous structural and functional characterizations of worm and mouse complexins have indicated the membrane curvature-sensing C-terminal domain of these proteins is responsible for differences in inhibitory function. We have characterized the structure and dynamics of the mCpx1 CTD in the absence and presence of membranes and membrane mimetics using NMR, ESR, and optical spectroscopies. In the absence of lipids, the mCpx1 CTD features a short helix near its N-terminus and is otherwise disordered. In the presence of micelles and small unilamellar vesicles, the mCpx1 CTD forms a discontinuous helical structure in its C-terminal 20 amino acids, with no preference for specific lipid compositions. In contrast, the mCpx1 CTD shows distinct compositional preferences in its interactions with large unilamellar vesicles. These studies identify structural divergences in the mCpx1 CTD relative to the wCpx1 CTD in regions that are known to be critical to the wCpx1 CTD's role in inhibiting spontaneous fusion of synaptic vesicles, suggesting a potential structural basis for evolutionary divergences in complexin function.
ABSTRACT: The sensitivity of magnetic resonance force microscopy (MRFM) is limited by surface noise. Coating a thin-film polymer sample with metal has been shown to decrease, by orders of magnitude, sample-related force noise and frequency noise in MRFM experiments. Using both MRFM and inductively detected measurements of electron-spin resonance, we show that thermally evaporating a 12 nm gold layer on a 40 nm nitroxide-doped polystyrene film inactivates the nitroxide spin labels to a depth of 20 nm, making single-spin measurements difficult or impossible. We introduce a "laminated sample" protocol in which the gold layer is first evaporated on a sacrificial polymer. The sample is deposited on the room-temperature gold layer, removed using solvent lift-off, and placed manually on a coplanar waveguide. Electron spin resonance (ESR) of such a laminated sample was detected via MRFM at cryogenic temperatures using a high-compliance cantilever with an integrated 100-nm-scale cobalt tip. A 20-fold increase of spin signal was observed relative to a thin-film sample prepared instead with an evaporated metal coating. The observed signal is still somewhat smaller than expected, and we discuss possible remaining sources of signal loss.
ABSTRACT: Bacterial chemoreceptors regulate the cytosolic multidomain histidine kinase CheA through largely unknown mechanisms. Residue substitutions in the peptide linkers that connect the P4 kinase domain to the P3 dimerization and P5 regulatory domain affect CheA basal activity and activation. To understand the role that these linkers play in CheA activity, the P3-to-P4 linker (L3) and P4-to-P5 linker (L4) were extended and altered in variants of Thermotoga maritima (Tm) CheA. Flexible extensions of the L3 and L4 linkers in CheA-LV1 (linker variant 1) allowed for a well-folded kinase domain that retained wild-type (WT)-like binding affinities for nucleotide and normal interactions with the receptor-coupling protein CheW. However, CheA-LV1 autophosphorylation activity registered ~50-fold lower compared to WT. Neither a WT nor LV1 dimer containing a single P4 domain could autophosphorylate the P1 substrate domain. Autophosphorylation activity was rescued in variants with extended L3 and L4 linkers that favor helical structure and heptad spacing. Autophosphorylation depended on linker spacing and flexibility and not on sequence. Pulse-dipolar electron-spin resonance (ESR) measurements with spin-labeled adenosine 5′-triphosphate (ATP) analogues indicated that CheA autophosphorylation activity inversely correlated with the proximity of the P4 domains within the dimers of the variants. Despite their separation in primary sequence and space, the L3 and L4 linkers also influence the mobility of the P1 substrate domains. In all, interactions of the P4 domains, as modulated by the L3 and L4 linkers, affect domain dynamics and autophosphorylation of CheA, thereby providing potential mechanisms for receptors to regulate the kinase.
ABSTRACT: Reduction of nitrite anions (NO2–) to nitric oxide (NO), nitrous oxide (N2O) and ultimately dinitrogen (N2) takes place in a variety of environments, including in the soil as part of the biogeochemical nitrogen cycle and in acidified nuclear waste. Nitrite reduction typically takes place within the coordination sphere of a redox-active transition metal. Here we show that Lewis acid coordination can substantially modify the reduction potential of this polyoxoanion to allow for its reduction under non-aqueous conditions (–0.74 V versus NHE). Detailed characterization confirms the formation of the borane-capped radical nitrite dianion (NO22–), which features a N(II) oxidation state. Protonation of the nitrite dianion results in the facile loss of nitric oxide (NO), whereas its reaction with NO results in disproportionation to nitrous oxide (N2O) and nitrite (NO2–). This system connects three redox levels in the global nitrogen cycle and provides fundamental insights into the conversion of NO2– to NO.
ABSTRACT: Cryptochrome (CRY) entrains the fly circadian clock by binding to Timeless (TIM) in light. Undocking of a helical C-terminal tail (CTT) in response to photoreduction of the CRY flavin cofactor gates TIM recognition. We present a generally applicable select western-blot-free tagged-protein interaction (SWFTI) assay that allowed the quantification of CRY binding to TIM in dark and light. The assay was used to study CRY variants with residue substitutions in the flavin pocket and correlate their TIM affinities with CTT undocking, as measured by pulse-dipolar ESR spectroscopy and evaluated by molecular dynamics simulations. CRY variants with the CTT removed or undocked bound TIM constitutively, whereas those incapable of photoreduction bound TIM weakly. In response to the flavin redox state, two conserved histidine residues contributed to a robust on/off switch by mediating CTT interactions with the flavin pocket and TIM. Our approach provides an expeditious means to quantify the interactions of difficult-to-produce proteins.
ABSTRACT: We describe the application of the microscopic-order-macroscopic-disorder (MOMD) approach, developed for the analysis of dynamic 2H NMR lineshapes in the solid state, to unravel interactions among the constituents of metal–organic frameworks (MOFs) that comprise mobile components. MOMD was applied recently to University of Windsor Dynamic Material (UWDM) MOFs with one mobile crown ether per cavity. In this work, we study UWDM-9-d4, which comprises a mobile 2H-labeled phenyl-ring residue along with an isotopically unlabeled 24C8 crown ether. We also study UiO-68-d4, which is structurally similar to UWDM-9-d4 but lacks the crown ether. The physical picture consists of the NMR probe–the C–D bonds of the phenyl-d4 rotor–diffusing locally (diffusion tensor R) in the presence of a local ordering potential, u. For UiO-68-d4, we find it sufficient to expand u in terms of four real Wigner functions, D0|K|L, overall 2–3 kT in magnitude, with R∥ relatively fast, and R⊥; in the (2.8–5.0) × 102 s-1 range. For UWDM-9-d4, u requires only two terms 2–3 kT in magnitude and slower rate constants R∥ and R⊥. In the more crowded macrocycle-containing UWDM-9-d4 cavity, phenyl-d4 dynamics is more isotropic and is described by a simpler ordering potential. This is ascribed to cooperative phenyl-ring/macrocycle motion, which yields a dynamic structure more uniform in character. The experimental 2H spectra used here were analyzed previously with a multi-simple-mode (MSM) approach where several independent simple motional modes are combined. Where possible, similar features have been identified and used to compare the two approaches.
ABSTRACT: We have developed the stochastic microscopic‐order‐macroscopic‐disorder (MOMD) approach for elucidating dynamic structures in the solid‐state from 2H NMR lineshapes. In MOMD, the probe experiences an effective/collective motional mode. The latter is described by a potential, u, which represents the local spatial‐restrictions, a local‐motional diffusion tensor, R, and key features of local geometry. Previously we applied MOMD to the well‐structured core domain of the 3‐fold‐symmetric twisted polymorph of the Aβ40‐amyloid fibril. Here, we apply it to the N‐terminal domain of this fibril. We find that the dynamic structures of the two domains are largely similar but differ in the magnitude and complexity of the key physical parameters. This interpretation differs from previous multisimple‐mode (MSM) interpretations of the same experimental data. MSM used for the two domains different combinations of simple motional modes taken to be independent. For the core domain, MOMD and MSM disagree on the character of the dynamic structure. For the N‐terminal domain, they even disagree on whether this chain segment is structurally ordered (MOMD finds that it is), and whether it undergoes a phase transition at 260 K where bulklike water located in the fibril matrix freezes (MOMD finds that it does not). These are major differences associated with an important system. While the MOMD description is a physically sound one, there are drawbacks in the MSM descriptions. The results obtained in this study promote our understanding of the dynamic structure of protein aggregates. Thus, they contribute to the effort to pharmacologically control neurodegenerative disorders believed to be caused by such aggregates.
ABSTRACT: Entry of coronaviruses into host cells is mediated by the viral spike protein. Previously, we identified the bona fide fusion peptides (FPs) for severe acute respiratory syndrome coronavirus ("SARS‐1") and severe acute respiratory syndrome coronavirus‐2 ("SARS‐2") using electron spin resonance spectroscopy. We also found that their FPs induce membrane ordering in a Ca2+‐dependent fashion. Here we study which negatively charged residues in SARS‐1 FP are involved in this binding, to build a topological model and clarify the role of Ca2+. Our systematic mutation study on the SARS‐1 FP shows that all six negatively charged residues contribute to the FP's membrane ordering activity, with D812 the dominant residue. The corresponding SARS‐2 residue D830 plays an equivalent role. We provide a topological model of how the FP binds Ca2+ ions: its two segments FP1 and FP2 each bind one Ca2+. The binding of Ca2+, the folding of FP (both studied by isothermal titration calorimetry experiments), and the ordering activity correlate very well across the mutants, suggesting that the Ca2+ helps the folding of FP in membranes to enhance the ordering activity. Using a novel pseudotyped viral particle‐liposome methodology, we monitored the membrane ordering induced by the FPs in the whole spike protein in its trimer form in real time. We found that the SARS‐1 and SARS‐2 pseudotyped viral particles also induce membrane ordering to the extent that separate FPs do, and mutations of the negatively charged residues also significantly suppress the membrane ordering activity. However, the slower kinetics of the FP ordering activity versus that of the pseudotyped viral particle suggest the need for initial trimerization of the FPs.
ABSTRACT: Environment-triggered protein conformational changes have garnered wide interest in both fundamental research, for deciphering in vivo acclimatory responses, and practical applications, for designing stimuli-responsive probes. Here, we propose a protein–chromophore regulatory mechanism that allows for manipulation of C-phycocyanin (C-PC) from Spirulina platensis by environmental pH and UV irradiation. Using small-angle X-ray scattering, a pH-mediated C-PC assembly–disassembly pathway, from monomers to nonamers, was unraveled. Such flexible protein matrices impart tunability to the embedded tetrapyrroles, whose photochemical behaviors were found to be modulated by protein assembly states. UV irradiation on C-PC triggers pH-dependent singlet oxygen (1O2) generation and conformational changes. Intermolecular photo-crosslinking occurs at pH 5.0 via dityrosine species, which bridges solution-based C-PC oligomers into unprecedented dodecamers and 24-mers. These supramolecular assemblies impart C-PC at pH 5.0, which significantly enhanced 1O2 yield, fluorescence, and photostability relative to those at other pH values, a finding that makes C-PC appealing for tumor-targeted photodynamic therapy.
ABSTRACT: During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane‐bound scaffold. Here, we found that highly basic membrane‐proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC‐membrane interactions and inhibit NEC‐mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane‐budding activity of the NEC to prevent capsid‐less budding. IMPORTANCE Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world's population. During replication, herpesviruses export their capsids from the nucleus into the cytoplasm by an unusual mechanism in which the viral nuclear egress complex (NEC) deforms the nuclear membrane around the capsid. However, how membrane deformation is achieved is unclear. Here, we show that the NEC from herpes simplex virus 1, a prototypical herpesvirus, uses clusters of positive charges to bind membranes and order membrane lipids. Reducing the positive charge or introducing negative charges weakens the membrane deforming ability of the NEC. We propose that the virus employs electrostatics to deform nuclear membrane around the capsid and can control this process by changing the NEC charge through phosphorylation. Blocking NEC-membrane interactions could be exploited as a therapeutic strategy.
ABSTRACT: CW saturation experiments are widely used in ESR studies of relaxation processes in proteins and lipids. We develop the theory of saturation in ESR spectra in terms of its close relation with that of 2D‐ELDOR. Our treatment of saturation is then based on the microscopic order macroscopic disorder (MOMD) model and can be used to fit the full CW saturation spectrum, rather than fitting just the peak–peak amplitude as a function of microwave field B1 as is commonly done. This requires fewer experiments to yield effects on T1, as well as provides a more extensive dynamic structural picture, for example, for scanning experiments on different protein sites. The code is released as a publicly available software package in Python that can be used to fit CW saturation spectra from biological samples of interest.
ABSTRACT: All radical S‐adenosylmethionine (radical‐SAM) enzymes, including the noncanonical radical‐SAM enzyme diphthamide biosynthetic enzyme Dph1–Dph2, require at least one [4Fe–4S](Cys)3 cluster for activity. It is well‐known in the radical‐SAM enzyme community that the [4Fe–4S](Cys)3 cluster is extremely air‐sensitive and requires strict anaerobic conditions to reconstitute activity in vitro. Thus, how such enzymes function in vivo in the presence of oxygen in aerobic organisms is an interesting question. Working on yeast Dph1–Dph2, we found that consistent with the known oxygen sensitivity, the [4Fe–4S] cluster is easily degraded into a [3Fe–4S] cluster. Remarkably, the small iron‐containing protein Dph3 donates one Fe atom to convert the [3Fe–4S] cluster in Dph1–Dph2 to a functional [4Fe–4S] cluster during the radical‐SAM enzyme catalytic cycle. This mechanism to maintain radical‐SAM enzyme activity in aerobic environments is likely general, and Dph3‐like proteins may exist to keep other radical‐SAM enzymes functional in aerobic environments.
ABSTRACT: Noise impedes experimental studies by reducing signal resolution and/or suppressing weak signals. Signal averaging and filtering are the primary methods used to reduce noise, but they have limited effectiveness and lack capabilities to recover signals at low signal‐to‐noise ratios (SNRs). We utilize a wavelet transform‐based approach to effectively remove noise from spectroscopic data. The wavelet denoising method we use is a significant improvement on standard wavelet denoising approaches. We demonstrate its power in extracting signals from noisy spectra on a variety of signal types ranging from hyperfine lines to overlapped peaks to weak peaks overlaid on strong ones, drawn from electron‐spin‐resonance spectroscopy. The results show that one can accurately extract details of complex spectra, including retrieval of very weak ones. It accurately recovers signals at an SNR of ˜1 and improves the SNR by about 3 orders of magnitude with high fidelity. Our examples show that one is now able to address weaker SNR signals much better than by previous methods. This new wavelet approach can be successfully applied to other spectroscopic signals.
ABSTRACT: Coronaviruses are a major infectious disease threat, and include the zoonotic‐origin human pathogens SARS‐CoV‐2, SARS‐CoV, and MERS‐CoV (SARS‐2, SARS‐1, and MERS). Entry of coronaviruses into host cells is mediated by the spike (S) protein. In our previous ESR studies, the local membrane ordering effect of the fusion peptide (FP) of various viral glycoproteins including the S of SARS‐1 and MERS has been consistently observed. We previously determined that the sequence immediately downstream from the S2′ cleavage site is the bona fide SARS‐1 FP. In this study, we used sequence alignment to identify the SARS‐2 FP, and studied its membrane ordering effect. Although there are only three residue differences, SARS‐2 FP induces even greater membrane ordering than SARS‐1 FP, possibly due to its greater hydrophobicity. This may be a reason that SARS‐2 is better able to infect host cells. In addition, the membrane binding enthalpy for SARS‐2 is greater. Both the membrane ordering of SARS‐2 and SARS‐1 FPs are dependent on Ca2+, but that of SARS‐2 shows a greater response to the presence of Ca2+. Both FPs bind two Ca2+ ions as does SARS‐1 FP, but the two Ca2+ binding sites of SARS‐2 exhibit greater cooperativity. This Ca2+ dependence by the SARS‐2 FP is very ion‐specific. These results show that Ca2+ is an important regulator that interacts with the SARS‐2 FP and thus plays a significant role in SARS‐2 viral entry. This could lead to therapeutic solutions that either target the FP‐calcium interaction or block the Ca2+ channel.
ABSTRACT: Two‐dimensional electron‐electron double resonance (2D‐ELDOR) provides extensive insight into molecular motions. Recent developments permitting experiments at higher frequencies (95 GHz) provide molecular orientational resolution, enabling a clearer description of the nature of the motions. In previous work, we provided simulations for the case of domain motions within proteins that are themselves slowly tumbling in a solution. In order to perform these simulations, it was found that the standard approach of solving the relevant stochastic Liouville equation using the efficient Lanczos algorithm for this case breaks down, so algorithms were employed that rely on the Arnoldi iteration. While they lead to accurate simulations, they are very time‐consuming. In this work, we focus on a variant known as the rational Arnoldi algorithm. We show that this can achieve a significant reduction in computation time. The stochastic Liouville matrix, which is of very large dimension, N, is first reduced to a much smaller dimension, m, e.g., from N ˜ O(104) to m ˜ 60, that spans the relevant Krylov subspace from which the spectrum is predicted. This requires the selection of the m frequency shifts to be utilized. A method of adaptive shift choice is introduced to optimize this selection. We also find that these procedures help in optimizing the pruning procedure that greatly reduces the dimension of the initial N dimensional stochastic Liouville matrix in such subsequent computations.
ABSTRACT: Electrons added to TiO2 and other semiconductors often occupy trap states, whose reactivity can determine the catalytic and stoichiometric chemistry of the material. We previously showed that reduced aqueous colloidal TiO2 nanoparticles have two distinct classes of thermally equilibrated trapped electrons, termed Red/e− and Blue/e−. Presented here are parallel optical and electron paramagnetic resonance (EPR) kinetic studies of the reactivity of these electrons with solution-based oxidants. Optical stopped-flow measurements monitoring the reactions of TiO2/e− with substoichiometric oxidants showed a surprising pattern: an initial fast (seconds) decrease in TiO2/e− absorbance followed by a secondary, slow (minutes) increase in the broad TiO2/e− optical feature. The analysis revealed that the fast decrease is due to the preferential oxidation of the Red/e− trap states and the slow increase results from the re-equilibration of electrons from Blue/e− to Red/e− states. This kinetic model was confirmed by freeze-quench EPR measurements. Quantitative analysis of the kinetic data demonstrated that Red/e− react ˜5 times faster than Blue/e− with the nitroxyl radical oxidant 4-methoxy-2,2,6,6-tetramethyl-1-piperidinyloxyl (4-MeO-TEMPO). Similar reactivity patterns were also observed in oxidations of TiO2/e− by O2, which like 4-MeO-TEMPO is a proton-coupled electron transfer (PCET) oxidant, and by the pure electron transfer (ET) oxidant potassium triiodide (KI3). This suggests that the faster intrinsic reactivity of one trap state over another on the seconds–minutes time scale is likely a general feature of reduced TiO2 reactivity. This differential trap-state reactivity is likely to influence the performance of TiO2 in photochemical/electrochemical devices, and it suggests an opportunity for tuning catalysis.
ABSTRACT: Exchange processes which include conformational change, protonation/deprotonation, and binding equilibria are routinely studied by 2D exchange NMR techniques, where information about the exchange of nuclei between environments with different NMR shifts is obtained from the development of cross‐peaks. Whereas 2D NMR enables the real time study of millisecond and slower exchange processes, 2D ESR in the form of 2D‐ELDOR (two‐dimensional electron‐electron double resonance) has the potential for such studies over the nanosecond to microsecond real time scales. Cross‐peak development due to chemical exchange has been seen previously for semiquinones in ESR, but this is not possible for most common ESR probes, such as nitroxides, studied at typical ESR frequencies because, unlike NMR, the exchanging states yield ESR signals that are not resolved from each other within their respective line widths. But at 95 GHz, it becomes possible to resolve them in many cases because of the increased g‐factor resolution. The 95 GHz instrumental developments occurring at ACERT now enable such studies. We demonstrate these new capabilities in two studies: (A) the protonation/deprotonation process for a pH‐sensitive imidazoline spin label in aqueous solution where the exchange rate and the population ratio of the exchanging states are controlled by the concentration and pH of the buffer solution, respectively, and (B) a nitroxide radical partitioning between polar (aqueous) and nonpolar (phospholipid) environments in multilamellar lipid vesicles, where the cross‐peak development arises from the exchange of the nitroxide between the two phases. This work represents the first example of the observation and analysis of cross‐peaks arising from chemical exchange processes involving nitroxide spin labels.
ABSTRACT: Bacterial chemoreceptors, the histidine kinase CheA, and the coupling protein CheW form transmembrane molecular arrays with remarkable sensing properties. The receptors inhibit or stimulate CheA kinase activity depending on the presence of attractants or repellants, respectively. We engineered chemoreceptor cytoplasmic regions to assume a trimer of receptor dimers configuration that formed well‐defined complexes with CheA and CheW and promoted a CheA kinase‐off state. These mimics of core signaling units were assembled to homogeneity and investigated by site‐directed spin‐labeling with pulse‐dipolar electron‐spin resonance spectroscopy (PDS), small‐angle x‐ray scattering, targeted protein cross‐linking, and cryo–electron microscopy. The kinase‐off state was especially stable, had relatively low domain mobility, and associated the histidine substrate and docking domains with the kinase core, thus preventing catalytic activity. Together, these data provide an experimentally restrained model for the inhibited state of the core signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.
Probing the Interaction between HIV-1 Protease and the Homodimeric p66/p66′ Reverse Transcriptase Precursor by Double Electron-Electron Resonance EPR Spectroscopy T. Schmidt, J. M. Louis, and G. M. Clore ChemBioChem21 (21), 3051-3055 (2020)
ABSTRACT: Following excision from the Gag-Pol polyprotein, HIV-1 reverse transcriptase is released as an asymmetric homodimer comprising two p66 subunits that are structurally dissimilar but identical in amino acid sequence. Subsequent cleavage of the RNase H domain from only one of the subunits, denoted p66′, results in the formation of the mature p66/p51 enzyme in which catalytic activity resides in the p66 subunit, and the p51 subunit (derived from p66′) provides a supporting structural scaffold. Here, we probe the interaction of the p66/p66′ asymmetric reverse transcriptase precursor with HIV-1 protease by pulsed Q-band double electron-electron resonance EPR spectroscopy to measure distances between nitroxide labels introduced at surface-engineered cysteine residues. The data suggest that the flexible, exposed linker between the RNaseH and connection domains in the open state of the p66′ subunit binds to the active site of protease in a configuration that is similar to that of extended peptide substrates.
ABSTRACT: Mix-and-inject serial crystallography is an emerging technique that utilizes X-ray free-electron lasers (XFELs) and microcrystalline samples to capture atomically detailed snapshots of biomolecules as they function. Early experiments have yielded exciting results; however, there are limited options to characterize reactions in crystallo in advance of the beamtime. Complementary measurements are needed to identify the best conditions and timescales for observing structural intermediates. Here, we describe the interface of XFEL compatible mixing injectors with rapid freeze-quenching and X-band EPR spectroscopy, permitting characterization of reactions in crystals under the same conditions as an XFEL experiment. We demonstrate this technology by tracking the reaction of azide with microcrystalline myoglobin, using only a fraction of the sample required for a mix-and-inject experiment. This spectroscopic method enables optimization of sample and mixer conditions to maximize the populations of intermediate states, eliminating the guesswork of current mix-and-inject experiments.
ABSTRACT: Human T-cell leukemia virus type 1 is an oncovirus that causes aggressive adult T-cell leukemia but is also responsible for severe neurodegenerative and endocrine disorders. Combatting HTLV-1 infections requires a detailed understanding of the viral mechanisms in the host. Therefore, in vitro studies of important virus-encoded proteins would be critical. Our focus herein is on the HTLV-1-encoded regulatory protein p13II, which interacts with the inner mitochondrial membrane, increasing its permeability to cations (predominantly potassium, K+). Thereby, this protein affects mitochondrial homeostasis. We report on our progress in developing specific protocols for heterologous expression of p13II in E. coli, and methods for its purification and characterization. We succeeded in producing large quantities of highly-pure full-length p13II, deemed to be its fully functional form. Importantly, our particular approach based on the fusion of ubiquitin to the p13II C-terminus was instrumental in increasing the persistently low expression of soluble p13II in its native form. We subsequently developed approaches for protein spin labeling and a conformation study using double electron-electron resonance (DEER) spectroscopy and a fluorescence-based cation uptake assay for p13II in liposomes. Our DEER results point to large protein conformation changes occurring upon transition from the soluble to the membrane-bound state. The functional assay on p13II-assisted transport of thallium (Tl+) through the membrane, wherein Tl+ substituted for K+, suggests transmembrane potential involvement in p13II function. Our study lays the foundation for expansion of in vitro functional and structural investigations on p13II and would aid in the development of structure-based protein inhibitors and markers.
SIGNIFICANCE: Cholesterol regulates critical cell functions, including lysis, viral budding, and antibiotic resistance, by modifying the bending rigidity of cell membranes; i.e., the ability of membranes to bend or withstand mechanical stresses. A molecular-level understanding of these functions requires knowledge of how cholesterol modifies membrane mechanics over relevant length and time scales. Currently, it is widely accepted that cholesterol has no effect on the mechanical properties of unsaturated lipid membranes, implying that viruses, for example, can bud from regions enriched in (poly)unsaturated lipids. Our observations that cholesterol causes local stiffening in DOPC membranes indicate that a reassessment of existing concepts is necessary. These findings have far-reaching implications in understanding cholesterol's role in biology and its applications in bioengineering and drug design.
ABSTRACT: Cholesterol is an integral component of eukaryotic cell membranes and a key molecule in controlling membrane fluidity, organization, and other physicochemical parameters. It also plays a regulatory function in antibiotic drug resistance and the immune response of cells against viruses, by stabilizing the membrane against structural damage. While it is well understood that, structurally, cholesterol exhibits a densification effect on fluid lipid membranes, its effects on membrane bending rigidity are assumed to be nonuniversal; i.e., cholesterol stiffens saturated lipid membranes, but has no stiffening effect on membranes populated by unsaturated lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). This observation presents a clear challenge to structure–property relationships and to our understanding of cholesterol-mediated biological functions. Here, using a comprehensive approach–combining neutron spin-echo (NSE) spectroscopy, solid-state deuterium NMR (2H NMR) spectroscopy, and molecular dynamics (MD) simulations–we report that cholesterol locally increases the bending rigidity of DOPC membranes, similar to saturated membranes, by increasing the bilayer's packing density. All three techniques, inherently sensitive to mesoscale bending fluctuations, show up to a threefold increase in effective bending rigidity with increasing cholesterol content approaching a mole fraction of 50%. Our observations are in good agreement with the known effects of cholesterol on the area-compressibility modulus and membrane structure, reaffirming membrane structure–property relationships. The current findings point to a scale-dependent manifestation of membrane properties, highlighting the need to reassess cholesterol's role in controlling membrane bending rigidity over mesoscopic length and time scales of important biological functions, such as viral budding and lipid–protein interactions.
ABSTRACT: The microscopic-order-macroscopic-disorder (MOMD) approach for NMR lineshape analysis has been applied to the University of Windsor Dynamic Materials (UWDM) of types 1, 2, α-3, β-3, and 5, which are metal–organic frameworks (MOFs) comprising mobile mechanically interlocked molecules (MIMs). The mobile MIM components are selectively deuterated crown ether macrocycles – 24C6, 22C6, and B24C6. Their motion is described in MOMD by an effective/collective dynamic mode characterized by a diffusion tensor, R, a restricting/ordering potential, u, expanded in the Wigner rotation matrix elements, D0,KL, and features of local geometry. Experimental 2H lineshapes are available over 220 K (on average) and in some cases 320 K. They are reproduced with axial R, u given by the terms D0,02 and D0,|2|2, and established local geometry. For UWDM of types 1, β-3, and 5, where the macrocycle resides in a relatively loose space, u is in the 1–3 kT, R∥ in the (1.0–2.5) × 106 s–1, and R⊥ in the (0.4–2.5) × 104 s–1 range; the deuterium atom is bonded to a carbon atom with tetrahedral coordination character. For UWDM of types 2 and α-3, where the macrocycle resides in a much tighter space, a substantial change in the symmetry of u and the coordination character of the 2H-bonded carbon are detected at higher temperatures. The activation energies for R∥ and R⊥ are characteristic of each system. The MOMD model is general; effective/collective dynamic modes are treated. The characteristics of motion, ordering, and geometry are physically well-defined; they differ from case to case in extent and symmetry but not in essence. Physical clarity and consistency provide new insights. A previous interpretation of the same experimental data used models consisting of collections of independent simple motions. These models are specific to each case and temperature. Within their scope, generating consistent physical pictures and comparing cases are difficult; possible collective modes are neglected.
ABSTRACT: Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homology, and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approximately 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.
IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging infectious disease with zoonotic potential and has reservoirs in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans, with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data have found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This information can guide therapeutic solutions to block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.
ABSTRACT: Two-dimensional electron-electron double resonance (2D-ELDOR) provides extensive insight into molecular motions. Recent developments permitting experiments at higher frequencies (95 GHz) provide molecular orientational resolution, enabling a clearer description of the nature of the motions. In this work, simulations are provided for the example of domain motions within proteins that are themselves slowly tumbling in solution. These show the nature of the exchange cross-peaks that are predicted to develop in real time from such domain motions. However, we find that the existing theoretical methods for computing 2D-ELDOR experiments over a wide motional range begin to fail seriously when applied to very slow motions characteristic of proteins in solution. One reason is the failure to obtain accurate eigenvectors and eigenvalues of the complex symmetric stochastic Liouville matrices describing the experiment when computed by the efficient Lanczos algorithm in the range of very slow motion. Another, perhaps more serious, issue is that these matrices are "non-normal," such that for the very slow motional range even rigorous diagonalization algorithms do not yield the correct eigenvalues and eigenvectors. We have employed algorithms that overcome both these issues and lead to valid 2D-ELDOR predictions even for motions approaching the rigid limit. They are utilized to describe the development of cross-peaks in 2D-ELDOR at 95 GHz for a particular case of domain motion.
ABSTRACT: RGD is a prolific example of a tripeptide used in biomaterials for cell adhesion, but the potency of free or surface-bound RGD tripeptide is orders-of-magnitude less than the RGD domain within natural proteins. We designed a set of peptides with varying lengths, composed of fragments of fibronectin protein whose central three residues are RGD, in order to vary their conformational behavior without changing the binding site's chemical environment. With these peptides, we measure the conformational dynamics and transient structure of the active site. Our studies reveal how flanking residues affect conformational behavior and integrin binding. We find that disorder of the binding site is important to the potency of RGD peptides and that transient hydrogen bonding near the RGD site affects both the energy landscape roughness of the peptides and peptide binding. This phenomenon is independent of longer-range folding interactions and helps explain why short binding sequences, including RGD itself, do not fully replicate the integrin-targeting properties of extracellular matrix proteins. Our studies reinforce that peptide binding is a holistic event and fragments larger than those directly involved in binding should be considered in the design of peptide epitopes for functional biomaterials.
ABSTRACT: Double electron-electron resonance (DEER) EPR spectroscopy is a powerful method for obtaining distance distributions between pairs of engineered nitroxide spin-labels in proteins and other biological macromolecules. These measurements require the use of cryogenic temperatures (77 K or less) to prolong the phase memory relaxation time (Tm) sufficiently to enable detection of a DEER echo curve. Generally, a cryoprotectant such as glycerol is added to protein samples to facilitate glass formation and avoid protein clustering (which can result in a large decrease in Tm) during relatively slow flash freezing in liquid N2. However, cryoprotectants are osmolytes and can influence protein folding/unfolding equilibria, as well as species populations in weak multimeric systems. Here we show that submillisecond rapid freezing, achieved by high velocity spraying of the sample onto a rapidly spinning, liquid nitrogen cooled copper disc obviates the requirement for cryoprotectants and permits high quality DEER data to be obtained in absence of glycerol. We demonstrate this approach on five different protein systems: protein A, the metastable drkN SH3 domain, urea-unfolded drkN SH3, HIV-1 reverse transcriptase, and the transmembrane domain of HIV-1 gp41 in lipid bicelles.
ABSTRACT: The self-assembly of short peptides gives rise to versatile nanoassemblies capable of promoting efficient catalysis. We have semi-rationally designed a series of seven-residue peptides that form hemin-binding catalytic amyloids to facilitate enantioselective cyclopropanation with efficiencies that rival those of engineered hemin proteins. These results demonstrate that: 1) Catalytic amyloids can bind complex metallocofactors to promote practically important multisubstrate transformations. 2) Even essentially flat surfaces of amyloid assemblies can impart a substantial degree of enantioselectivity without the need for extensive optimization. 3) The ease of peptide preparation allows for straightforward incorporation of unnatural amino acids and the preparation of peptides made from D-amino acids with complete reversal of enantioselectivity.
ABSTRACT: Ebola virus disease is a serious global health concern given its periodic occurrence, high lethality, and the lack of approved therapeutics. Certain drugs that alter intracellular calcium, particularly in endolysosomes, have been shown to inhibit Ebola virus infection; however, the underlying mechanism is unknown. Here, we provide evidence that Zaire ebolavirus (EBOV) infection is promoted in the presence of calcium as a result of the direct interaction of calcium with the EBOV fusion peptide (FP). We identify the glycoprotein residues D522 and E540 in the FP as functionally critical to EBOV's interaction with calcium. We show using spectroscopic and biophysical assays that interactions of the fusion peptide with Ca2+directly targets the Ebola virus fusion peptide and influences its conformation. As these residues are highly conserved across the Filoviridae, calcium's impact on fusion, and subsequently infectivity, is a key interaction that can be leveraged for developing strategies to defend against Ebola infection. This mechanistic insight provides a rationale for the use of calcium-interfering drugs already approved by the FDA as therapeutics against Ebola and enables further development of novel drugs to combat the virus.
ABSTRACT: Transient tyrosine and tryptophan radicals play key roles in the electron transfer (ET) reactions of photosystem (PS) II, ribonucleotide reductase (RNR), photolyase, and many other proteins. However, Tyr and Trp are not functionally interchangeable, and the factors controlling their reactivity are often unclear. Cytochrome c peroxidase (CcP) employs a Trp191⋅+ radical to oxidize reduced cytochrome c (Cc). Although a Tyr191 replacement also forms a stable radical, it does not support rapid ET from Cc. Here we probe the redox properties of CcP Y191 by non-natural amino acid substitution, altering the ET driving force and manipulating the protic environment of Y191. Higher potential fluorotyrosine residues increase ET rates marginally, but only addition of a hydrogen bond donor to Tyr191⋅ (via Leu232His or Glu) substantially alters activity by increasing the ET rate by nearly 30-fold. ESR and ESEEM spectroscopies, crystallography, and pH-dependent ET kinetics provide strong evidence for hydrogen bond formation to Y191⋅ by His232/Glu232. Rate measurements and rapid freeze quench ESR spectroscopy further reveal differences in radical propagation and Cc oxidation that support an increased Y191⋅ formal potential of ∼200 mV in the presence of E232. Hence, Y191 inactivity results from a potential drop owing to Y191⋅+ deprotonation. Incorporation of a well-positioned base to accept and donate back a hydrogen bond upshifts the Tyr⋅ potential into a range where it can effectively oxidize Cc. These findings have implications for the YZ/YD radicals of PS II, hole-hopping in RNR and cryptochrome, and engineering proteins for long-range ET reactions.
ABSTRACT: Metalloenzymes often utilize radicals in order to facilitate chemical reactions. Recently, DeGrado and co-workers have discovered that model proteins can efficiently stabilize semiquinone radical anion produced by oxidation of 3,5-di-tert-butylcatechol (DTBC) in the presence of two zinc ions. Here, we show that the number and the nature of metal ions have relatively minor effect on semiquinone stabilization in model proteins, with a single metal ion being sufficient for radical stabilization. The radical is stabilized by both metal ion, hydrophobic sequestration, and interactions with the hydrophilic residues in the protein interior resulting in a remarkable, nearly 500 mV change in the redox potential of the SQ.–/catechol couple compared to bulk aqueous solution. Moreover, we have created 4G-UFsc, a single metal ion-binding protein with pm affinity for zinc that is higher than any other reported model systems and is on par with many natural zinc-containing proteins. We expect that the robust and easy-to-modify DFsc/UFsc family of proteins will become a versatile tool for mechanistic model studies of metalloenzymes.
ABSTRACT: We report a facile synthetic approach to create stable radical block copolymers containing a secondary fluorinated block via anionic polymerization using a bulky, sterically hindered countercation composed of a sodium ion and di-benzo-18-crown-6 complex. The synthetic conditions described in this report allowed for controlled molecular weights and dispersity (<1.3) of both homopolymers: poly(2,2,6,6-tetramethyl-1-piperidinyloxy-methacrylate) (PTMA) and poly(2,2,2-trifluoroethyl methacrylate) (PTFEMA) as well as their block copolymers (PTMA-b-PTFEMA). The stable radical concentration of the polymers was determined by electron spin resonance (ESR) and showed radical content above 70%. An analysis of the microphase morphologies in PTMA-b-PTFEMA thin films via atomic force microscopy (AFM) and grazing incidence small angle X-ray scattering (GISAXS) showed clear evidence of long-range ordering of lamellar and cylindrical morphologies with 32 and 36 nm spacing, respectively. The long-range ordering of the morphologies was developed with the aid of two separate neutral layers: PTMA-ran-PTFEMA-ran-poly(hydroxyl ethyl methacrylate) (PHEMA) and poly(isobutyl methacrylate) (PiBMA)-ran-PTFEMA-ran-PHEMA, which helped us corroborate, along with the Zisman method, the surface energy estimation of PTMA to be 30.1 mJ m–2.
ABSTRACT: Magnetic resonance methods have been used extensively for over 50 years to elucidate molecular structure and dynamics of liquid crystals (LCs), providing information quite unique in its rigour and extent. The ESR- or NMR-active probe is often a solute molecule reporting on characteristics associated with the surrounding (LC) medium, which exerts the spatial restrictions on the probe. The theoretical approaches developed for LCs are applicable to anisotropic media in general. Of particular interest is the interior space of a globular protein labelled, e.g. with a nitroxide moiety or a 15N–1H bond. The ESR or NMR label plays the role of the probe and the internal protein surroundings the role of the anisotropic medium. A general feature of the restricted motions is the local ordering, i.e. the nature, magnitude and symmetry of the spatial restraints exerted at the site of the moving probe. This property is the main theme of the present review article. We outline its treatment in our work from both the theoretical and the experimental points of view, highlighting the new physical insights gained. Our illustrations include studies on thermotropic (nematic and smectic) and lyotropic liquid crystals formed by phospholipids, in addition to studies of proteins.
ABSTRACT: Ammonia oxidizing bacteria (AOB) use the cytotoxic, energetic molecule hydroxylamine (NH2OH) as a source of reducing equivalents for cellular respiration. Despite disproportionation or violent decomposition being typical outcomes of reactions of NH2OH with iron, AOB and anammox heme P460 proteins including cytochrome (cyt) P460 and hydroxylamine oxidoreductase (HAO) effect controlled, stepwise oxidation of NH2OH to nitric oxide (NO). Curiously, a recently characterized cyt P460 variant from the AOB Nitrosomonas sp. AL212 is able to form all intermediates of cyt P460 catalysis, but is nevertheless incompetent for NH2OH oxidation. We now show via site-directed mutagenesis, activity assays, spectroscopy, and structural biology that this lack of activity is attributable to the absence of a critical basic glutamate residue in the distal pocket above the heme P460 cofactor. This substitution is the only distinguishing characteristic of a protein that is otherwise effectively structurally and spectroscopically identical to an active variant. This highlights and reinforces a fundamental principal of metalloenzymology: metallocofactor inner-sphere geometric and electronic structures are in many cases insufficient for imbuing reactivity; a precisely defined outer coordination sphere contributed by the polypeptide matrix can be the key differentiator between a metalloenzyme and an unreactive metalloprotein.
ABSTRACT: pH is an important factor that affects the protein structure, stability, and activity. Here, we probe the nature of the low-pH structural form of the homodimeric CcdB (controller of cell death B) protein. Characterization of CcdB protein at pH 4 and 300 K using circular dichroism spectroscopy, 8-anilino-1-naphthalene-sulphonate binding, and Trp solvation studies suggests that it forms a partially unfolded state with a dry core at equilibrium under these conditions. CcdB remains dimeric at pH 4 as shown by multiple techniques, such as size-exclusion chromatography coupled to multiangle light scattering, analytical ultracentrifugation, and electron paramagnetic resonance. Comparative analysis using two-dimensional 15N-1H heteronuclear single-quantum coherence NMR spectra of CcdB at pH 4 and 7 suggests that the pH 4 and native state have similar but nonidentical structures. Hydrogen-exchange-mass-spectrometry studies demonstrate that the pH 4 state has substantial but anisotropic changes in local stability with core regions close to the dimer interface showing lower protection but some other regions showing higher protection relative to pH 7.
SIGNIFICANCE: All living things must sense and react to their environment. Many single-celled organisms do so by using two-component systems, most simply consisting of a sensor histidine kinase and a response regulator. These systems are involved in pathogenicity pathways and can be targeted by new antibiotics. However, the molecular mechanisms used by histidine kinases to translate sensing into responses are not well understood. To probe this general question, we apply a combination of biophysical techniques to a monomeric histidine kinase that senses blue light to determine the structural changes occurring upon activation. We find these changes to be similar to those predicted for the common dimeric histidine kinases, illustrating that the mechanism of activation is conserved regardless of oligomeric state.
ABSTRACT: Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light–sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme's residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.
SIGNIFICANCE: The tau protein is involved in Alzheimer's and other neurodegenerative diseases, where the location, morphology, and quantity of amyloid fibrils composed of tau correlate with the disease type and stage. While tau fibrillary aggregates have been colocalized in brains together with several cofactors, their role in fibril formation, structure, and seeding has been largely neglected. We show that seeding of tau aggregation is facilitated by polyanionic cofactors, and that seeded or recombinant mature fibrils depolymerize into monomers when their cofactor is removed. We show that cofactor-assisted seeding with mouse brain-derived tau fibrils yielded tau fibrils with distinct and narrowed structural properties compared with heparin-induced fibrils, suggesting that the fibrillar templates tuned the structure of the seeded fibril.
ABSTRACT: Amyloid fibrils are cross-β–rich aggregates that are exceptionally stable forms of protein assembly. Accumulation of tau amyloid fibrils is involved in many neurodegenerative diseases, including Alzheimer's disease (AD). Heparin-induced aggregates have been widely used and assumed to be a good tau amyloid fibril model for most biophysical studies. Here we show that mature fibrils made of 4R tau variants, prepared with heparin or RNA, spontaneously depolymerize and release monomers when their cofactors are removed. We demonstrate that the cross-β-sheet assembly formed in vitro with polyanion addition is unstable at room temperature. We furthermore demonstrate high seeding capacity with transgenic AD mouse brain-extracted tau fibrils in vitro that, however, is exhausted after one generation, while supplementation with RNA cofactors resulted in sustained seeding over multiple generations. We suggest that tau fibrils formed in brains are supported by unknown cofactors and inhere higher-quality packing, as reflected in a more distinct conformational arrangement in the mouse fibril-seeded, compared with heparin-induced, tau fibrils. Our study suggests that the role of cofactors in tauopathies is a worthy focus of future studies, as they may be viable targets for diagnosis and therapeutics.
ABSTRACT: This paper is a continuation of the method introduced by Srivastava and Freed (2017) that is a new method based on truncated singular value decomposition (TSVD) for obtaining physical results from experimental signals without any need for Tikhonov regularization or other similar methods that require a regularization parameter. We show here how to estimate the uncertainty in the SVD-generated solutions. The uncertainty in the solution may be obtained by finding the minimum and maximum values over which the solution remains converged. These are obtained from the optimum range of singular value contributions, where the width of this region depends on the solution point location (e.g., distance) and the signal-to-noise ratio (SNR) of the signal. The uncertainty levels typically found are very small with substantial SNR of the (denoised) signal, emphasizing the reliability of the method. With poorer SNR, the method is still satisfactory but with greater uncertainty, as expected. Pulsed dipolar electron spin resonance spectroscopy experiments are used as an example, but this TSVD approach is general and thus applicable to any similar experimental method wherein singular matrix inversion is needed to obtain the physically relevant result. We show that the Srivastava–Freed TSVD method along with the estimate of uncertainty can be effectively applied to pulsed dipolar electron spin resonance signals with SNR > 30, and even for a weak signal (e.g., SNR ≈ 3) reliable results are obtained by this method, provided the signal is first denoised using wavelet transforms (WavPDS).
ABSTRACT: Site-directed spin labeling of proteins by chemical modification of engineered cysteine residues with the molecule MTSSL (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl methanethiosulfonate) has been an invaluable tool for conducting double electron electron resonance (DEER) spectroscopy experiments. However, this method is generally limited to recombinant proteins with a limited number of reactive Cys residues that when modified will not impair protein function. Here, we present a method that allows for spin labeling of protein-nucleotide-binding sites by adenosine diphosphate (ADP) modified with a nitroxide moiety on the β-phosphate (ADP-β-S-SL). The synthesis of this ADP analog is straightforward and isolation of pure product is readily achieved on a standard reverse-phase high-performance liquid chromatography (HPLC) system. Furthermore, analyses of isolated ADP-β-S-SL by LC–mass spectrometry confirm that the molecule is very stable under ambient conditions. The crystal structure of ADP-β-S-SL bound to the ATP pocket of the histidine kinase CheA reveals specific targeting of the probe, whose nitroxide moiety is mobile on the protein surface. Continuous wave and pulsed-ESR measurements demonstrate the capability of ADP-β-S-SL to report on active site environment and provide reliable DEER distance constraints.
ABSTRACT: The dynamic nuclear polarization (DNP) efficiency is critically dependent on the properties of the radical, solvent, and solute constituting the sample system. In this study, we focused on the three spin e–e–n cross effect (CE)'s influence on the nuclear longitudinal relaxation time constant T1n, the build-up time constants of nuclear magnetic resonance (NMR) signal, TDNP and DNP-enhancement of NMR signal. The dipolar interaction strength between the electron spins driving the e–e–n process was systematically modulated using mono-, di-, tri-, and dendritic-nitroxide radicals, while maintaining a constant global electron spin concentration of 10 mM. Experimental results showed that an increase in electron spin clustering led to an increased electron spin depolarization, as mapped by electron double resonance (ELDOR), and a dramatically shortened T1n and TDNP time constants under static and magic angle spinning (MAS) conditions. A theoretical analysis reveals that strong e–e interactions, caused by electron spin clustering, increase the CE rate. The three spin e–e–n CE is a hitherto little recognized mechanism for shortening T1n and TDNP in solid-state NMR experiments at cryogenic temperatures, and offers a design principle to enhance the effective CE DNP enhancement per unit time. Fast CE rates will benefit DNP at liquid helium temperatures, or at higher magnetic fields and pulsed DNP, where slow e–e–n polarization transfer rate is a key bottleneck to achieving maximal DNP performance.
ABSTRACT: Alkyl chlorides are common functional groups in synthetic organic chemistry. However, the engagement of unactivated alkyl chlorides, especially tertiary alkyl chlorides, in transition-metal-catalyzed C–C bond formation remains challenging. Herein, we describe the development of a TiIII-catalyzed radical addition of 2° and 3° alkyl chlorides to electron-deficient alkenes. Mechanistic data are consistent with inner-sphere activation of the C–Cl bond featuring TiIII-mediated Cl atom abstraction. Evidence suggests that the active TiIII catalyst is generated from the TiIV precursor in a Lewis-acid-assisted electron transfer process.
ABSTRACT: Pulsed dipolar electron spin resonance spectroscopy (PDS) is a powerful tool for measuring distances in solution-state macromolecules. Paramagnetic metal ions, such as Cu2+, are used as spin probes because they can report on metalloprotein features and can be spectroscopically distinguished from traditional nitroxide (NO)-based labels. Here, we demonstrate site-specific incorporation of Cu2+ into non-metalloproteins through the use of a genetically encodable non-natural amino acid, 3-pyrazolyltyrosine (PyTyr). We first incorporate PyTyr in cyan fluorescent protein to measure Cu2+-to-NO distances and examine the effects of solvent conditions on Cu2+ binding and protein aggregation. We then apply the method to characterize the complex formed by the histidine kinase CheA and its target response regulator CheY. The X-ray structure of CheY–PyTyr confirms Cu labeling at PyTyr but also reveals a secondary Cu site. Cu2+-to-NO and Cu2+-to-Cu2+ PDS measurements of CheY–PyTyr with nitroxide-labeled CheA provide new insights into the conformational landscape of the phosphotransfer complex and have implications for kinase regulation.
ABSTRACT: We report a mild and efficient electrochemical protocol to access a variety of vicinally C–O and C–N difunctionalized compounds from simple alkenes. Detailed mechanistic studies revealed a distinct reaction pathway from those previously reported for TEMPO-mediated reactions. In this mechanism, electrochemically generated oxoammonium ion facilitates the formation of azidyl radical via a charge-transfer complex with azide, TEMPO–N3. DFT calculations together with spectroscopic characterization provided a tentative structural assignment of this charge-transfer complex. Kinetic and kinetic isotopic effect studies revealed that reversible dissociation of TEMPO–N3 into TEMPO⋅ and azidyl precedes the addition of these radicals across the alkene in the rate-determining step. The resulting azidooxygenated product could then be easily manipulated for further synthetic elaborations. The discovery of this new reaction pathway mediated by the TEMPO+/TEMPO⋅ redox couple may expand the scope of aminoxyl radical chemistry in synthetic contexts.
ABSTRACT: We have developed the microscopic-order-macroscopic-disorder (MOMD) approach for studying internal mobility in polycrystalline proteins with 2H lineshape analysis. The motion itself is expressed by a diffusion tensor, R, the local spatial restraints by a potential, u, and the "local geometry" by the relative orientation of the model-related and nuclear magnetic resonance-related tensors. Here, we apply MOMD to phenyl-ring dynamics in several Αβ40-amyloid-fibrils, and the villin headpiece subdomain (HP36). Because the available data are limited in extent and sensitivity, we adjust u and R in the relevant parameter ranges, fixing the "local geometry" in accordance with standard stereochemistry. This yields a physically well-defined and consistent picture of phenyl-ring dynamics, enabling comparison between different systems. In the temperature range of 278–308 K, u has a strength of (1.7–1.8) kT and a rhombicity of (2.4–2.6) kT, and R has components of 5.0 × 102 ≤ R⊥ ≤ 2.0 × 103 s–1 and 6.3 × 105 ≤ R∥ ≤ 2.0 × 106 s–1. At 278 K, fibril hydration increases the axiality of both u and R; HP36 hydration has a similar effect at 295 K, reducing R⊥ considerably. The D23N mutation slows down the motion of the probe; Αβ40 polymorphism affects both this motion and the related local potential. The present study identifies the impact of various factors on phenyl-ring mobility in amyloid fibrils and globular proteins; the difference between the two protein forms is considerable. The distinctive impact of hydration on phenyl-ring motion and previously studied methyl-group motion is also examined. The 2H lineshapes considered here were analyzed previously with various multi-simple-mode (MSM) models, where several simple motional modes are combined. The MOMD and MSM interpretations differ in essence.
ABSTRACT: An intensively investigated intermediate state of protein folding is the molten globule (MG) state, which contains secondary but hardly any tertiary structure. In previous work, we have determined the distances between interacting spins within maltose binding protein (MBP) in its native state using continuous wave and double electron–electron resonance (DEER) electron paramagnetic resonance (EPR) spectroscopy. Seven double mutants had been employed to investigate the structure within the two domains of MBP. DEER data nicely corroborated the previously available X-ray data. Even in its MG state, MBP is known to still bind its ligand maltose. We therefore hypothesized that there must be a defined structure around the binding pocket of MBP already in the absence of tertiary structure. Here we have investigated the functional and structural difference between native and MG state in the open and closed form with a new set of MBP mutants. In these, the spin-label positions were placed near the active site. Binding of its ligands leads to a conformational change from open to closed state, where the two domains are more closely together. The complete set of MBP mutants was analyzed at pH 3.2 (MG) and pH 7.4 (native state) using double-quantum coherence EPR. The values were compared with theoretical predictions of distances between the labels in biradicals constructed by molecular modeling from the crystal structures of MBP in open and closed form and were found to be in excellent agreement. Measurements show a defined structure around the binding pocket of MBP in MG, which explains maltose binding. A new and important finding is that in both states ligand-free MBP can be found in open and closed form, while ligand-bound MBP appears only in closed form because of maltose binding.
ABSTRACT: Membrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a homomeric protein can be independently modified for functional or spectroscopic studies. Here, we describe a general approach for in vitro assembly that can be used for the generation of heteromeric variants of homomeric membrane proteins. We establish the approach using GltPh, a glutamate transporter homolog that is trimeric in the native state. We use heteromeric GltPh transporters to directly demonstrate the lack of coupling in substrate binding and demonstrate how heteromeric transporters considerably simplify the application of DEER spectroscopy. Further, we demonstrate the general applicability of this approach by carrying out the in vitro assembly of VcINDY, a Na+-coupled succinate transporter and CLC-ec1, a Cl-/H+ antiporter.
ABSTRACT: Viral fusogens merge viral and cell membranes during cell penetration. Their ectodomains drive fusion by undergoing large-scale refolding, but little is known about the functionally important regions located within or near the membrane. Here we report the crystal structure of full-length glycoprotein B (gB), the fusogen from herpes simplex virus, complemented by electron spin resonance measurements. The membrane-proximal (MPR), transmembrane (TMD), and cytoplasmic (CTD) domains form a uniquely folded trimeric pedestal beneath the ectodomain, which balances dynamic flexibility with extensive, stabilizing membrane interactions. The postfusion conformation of the ectodomain suggests that the CTD likewise adopted the postfusion form. However, hyperfusogenic mutations, which destabilize the prefusion state of gB, target key interfaces and structural motifs that reinforce the observed CTD structure. Thus, a similar CTD structure must stabilize gB in its prefusion state. Our data suggest a model for how this dynamic, membrane-dependent 'clamp' controls the fusogenic refolding of gB.
ABSTRACT: The microscopic-order-macroscopic-disorder (MOMD) approach for 2H NMR line shape analysis is applied to dry and hydrated 3-fold- and 2-fold-symmetric amyloid-Aβ40 fibrils and protofibrils of the D23N mutant. The methyl moieties of L17, L34, V36 (C–CD3), and M35 (S–CD3) serve as probes. Experimental 2H spectra acquired previously in the 147–310 K range are used. MOMD describes local probe motion as axial diffusion (R tensor) in the presence of a potential, u, which represents the spatial restrictions exerted by the molecular surroundings. We find that R∥ = (0.2–3.3) × 104 s–1, R⊥ = (2.2–2.5) × 102 s–1, and R is tilted from the 2H quadrupolar tensor at 60–75°. The strength of u is in the (2.0–2.4) kT range; its rhombicity is substantial. The only methyl moieties affected by fibril hydration are those of M35, located at fibril interfaces. The associated local potentials change form abruptly around 260 K, where massive water freezing occurs. An independent study revealed unfrozen "tightly-peptide-bound" water residing at the interfaces of the 3-fold-symmetric Aβ40 fibrils and at the interfaces of the E22G and E22Δ Aβ40-mutant fibrils. Considering this to be the case in general for Aβ40-related fibrils, the following emerges. The impact of water freezing is transmitted selectively to the fibril structure through interactions with tightly-peptide-bound water, in this case of M35 methyl moieties. The proof that such waters reside at the interfaces of the 2-fold-symmetric fibril, and the protofibril of the D23N mutant, is new. MOMD provides information on the surroundings of the NMR probe directly via the potential, u, which is inherent to the model; a prior interpretation of the same experimental data does so partially and indirectly (see below). Thus, MOMD analysis of NMR line shapes as applied to amyloid fibrils/protein aggregates emerges as a consistent new tool for elucidating the properties of, and processes associated with, molecular environments in the fibril.
ABSTRACT: Diphthamide biosynthesis involves a carbon-carbon bond-forming reaction catalyzed by a radical S-adenosylmethionine (SAM) enzyme that cleaves a carbon-sulfur (C–S) bond in SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. Using rapid freezing, we have captured an organometallic intermediate with an iron-carbon (Fe–C) bond between ACP and the enzyme's [4Fe-4S] cluster. In the presence of the substrate protein, elongation factor 2, this intermediate converts to an organic radical, formed by addition of the ACP radical to a histidine side chain. Crystal structures of archaeal diphthamide biosynthetic radical SAM enzymes reveal that the carbon of the SAM C–S bond being cleaved is positioned near the unique cluster Fe, able to react with the cluster. Our results explain how selective C–S bond cleavage is achieved in this radical SAM enzyme.
ABSTRACT: We report on a new approach to the analysis of dynamic NMR lineshapes from polycrystalline (i.e., macroscopically disordered) samples in the presence of Magic Angle Spinning (MAS). This is an application of the Stochastic Liouville Equation developed by Freed and co-workers for treating restricted (i.e., microscopically ordered) motions. The 2H nucleus in an internally-mobile C–CD3 moiety serves as a prototype probe. The acronym is 2H/MOMD/MAS, where MOMD stands for "microscopic-order-macroscopic-disorder." The key elements describing internal motions – their type, the local spatial restrictions, and related features of local geometry – are treated in MOMD generally, within their rigorous three-dimensional tensorial requirements. Based on this representation a single physically well-defined model of local motion has the capability of reproducing experimental spectra. There exist other methods for analyzing dynamic 2H/MAS spectra which advocate simple motional modes. Yet, to reproduce satisfactorily the experimental lineshapes, one has either to use unusual parameter values, or combine several simple motional modes. The multi-simple-mode reasoning assumes independence of the constituent modes, features ambiguity as different simple modes may be used, renders inter-system comparison difficult as the overall models differ, and makes possible model-improvement only by adding yet another simple mode, i.e., changing the overall model. 2H/MOMD/MAS is free of such limitations and inherently provides a clear physical interpretation. These features are illustrated. The advantage of 2H/MOMD/MAS in dealing with sensitive but hardly investigated slow-motional lineshapes is demonstrated by applying it to actual experimental data. The results differ from those obtained previously with a two-site exchange scheme that yielded unusual parameters.
ABSTRACT: Coronaviruses (CoVs) are a major infectious disease threat and include the pathogenic human pathogens of zoonotic origin: severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV). Entry of CoVs into host cells is mediated by the viral spike (S) protein, which is structurally categorized as a class I viral fusion protein, within the same group as influenza virus and HIV. However, S proteins have two distinct cleavage sites that can be activated by a much wider range of proteases. The exact location of the CoV fusion peptide (FP) has been disputed. However, most evidence suggests that the domain immediately downstream of the S2′ cleavage site is the FP (amino acids 798–818 SFIEDLLFNKVTLADAGFMKQY for SARS-CoV, FP1). In our previous electron spin resonance spectroscopic studies, the membrane-ordering effect of influenza virus, HIV, and Dengue virus FPs has been consistently observed. In this study, we used this effect as a criterion to identify and characterize the bona fide SARS-CoV FP. Our results indicate that both FP1 and the region immediately downstream (amino acids 816–835 KQYGECLGDINARDLICAQKF, FP2) induce significant membrane ordering. Furthermore, their effects are calcium dependent, which is consistent with in vivo data showing that calcium is required for SARS-CoV S-mediated fusion. Isothermal titration calorimetry showed a direct interaction between calcium cations and both FPs. This Ca2+-dependency membrane ordering was not observed with influenza FP, indicating that the CoV FP exhibits a mechanistically different behavior. Membrane-ordering effects are greater and penetrate deeper into membranes when FP1 and FP2 act in a concerted manner, suggesting that they form an extended fusion "platform."
ABSTRACT: There are conflicting reports in the literature about the presence of room temperature conductivity in poly(2,2,6,6-tetramethylpiperidinyloxy methacrylate) (PTMA), a redox active polymer with radical groups pendent to an insulating backbone. To understand the variability in the findings across the literature and synthetic methods, we prepared PTMA using three living methods – anionic, ATRP and RAFT polymerization. We find that all three synthetic methods produce PTMA with radical yields of 70–80%, controlled molecular weight, and low dispersity. Additionally, we used on-chip EPR to probe the robustness of radical content in solid films under ambient air and light, and found negligible change in the radical content over time. Electrically, we found that PTMA is highly insulating – conductivity in the range 10–11 S cm–1 – regardless of the synthetic method of preparation. These findings provide greater clarity for potential applications of PTMA in energy storage.
The Eponymous Cofactors in Cytochrome P460s from Ammonia-Oxidizing Bacteria Are Iron Porphyrinoids Whose Macrocycles Are Dibasic M. A. Smith, and K. M. Lancaster Biochemistry57 (3), 334-343 (2018)
ABSTRACT: The enzymes hydroxylamine oxidoreductase and cytochrome (cyt) P460 contain related unconventional "heme P460" cofactors. These cofactors are unusual in their inclusion of nonstandard cross-links between amino acid side chains and the heme macrocycle. Mutagenesis studies performed on the Nitrosomonas europaea cyt P460 that remove its lysine–heme cross-link show that the cross-link is key to defining the spectroscopic properties and kinetic competence of the enzyme. However, exactly how this cross-link confers these features remains unclear. Here we report the 1.45 Å crystal structure of cyt P460 from Nitrosomonas sp. AL212 and conclude that the cross-link does not lead to a change in hybridization of the heme carbon participating in the cross-link but rather enforces structural distortions to the macrocycle away from planarity. Time-dependent density functional theory coupled to experimental structural and spectroscopic analysis suggest that this geometric distortion is sufficient to define the spectroscopic properties of the heme P460 cofactor and provide clues toward establishing a relationship between heme P460 electronic structure and function.
ABSTRACT: A range of powerful techniques based on pulse EPR spectroscopies is used extensively for measuring electron spin dipolar couplings between native or introduced paramagnetic reporters, from which accurate distances, and in certain cases molecular orientations, can be determined to aid in solving biomolecular structures. Notably, several versatile pulse EPR methods of high sensitivity were developed over the past two decades. The two main groups of pulse methods are represented by single- and double-resonance techniques, which together constitute pulse dipolar EPR spectroscopy (PDS). We describe the first group by outlining the basic principles and illustrate key techniques with several examples from the literature, as well as some newly produced for purposes of this chapter. Specifically, we describe double-quantum coherence (DQC) methods for robust selection of the dipolar coupling by filtering out undesired spin coherence pathways via phase cycling, and we also discuss single-resonance methods such as four- and five-pulse single-quantum coherence (SQC) methods, all of which benefit from maximizing the spectral coverage. The common features and conceptual differences between single- and double-resonance methods are discussed. In addition, the extensions of modern DQC and SQC methods to two-dimensional correlation spectroscopy for eliciting orientational information are included. Other types of 'single-frequency' PDS methods, not all of which are based on principles behind fully coherent single-resonance methods, are also discussed to show the diversity of PDS methods.
ABSTRACT: Ammonia (NH3)-oxidizing bacteria (AOB) derive total energy for life from the multi-electron oxidation of NH3 to nitrite (NO2−). One obligate intermediate of this metabolism is hydroxylamine (NH2OH), which can be oxidized to the potent greenhouse agent nitrous oxide (N2O) by the AOB enzyme cytochrome (cyt) P460. We have now spectroscopically characterized a 6-coordinate (6c) {FeNO}7 intermediate on the NH2OH oxidation pathway of cyt P460. This species has two fates: it can either be oxidized to the {FeNO}6 that then undergoes attack by NH2OH to ultimately generate N2O, or it can lose its axial His ligand, thus generating a stable, off-pathway 5-coordinate (5c) {FeNO}7 species. We show that the wild type (WT) cyt P460 exhibits a slow nitric oxide (NO)-independent conversion (kHis-off = 2.90 × 10−3 s−1), whereas a cross-link-deficient Lys70Tyr cyt P460 mutant protein underwent His dissociation via both a NO-independent (kHis-off = 3.8 × 10−4 s−1) and a NO-dependent pathway [kHis-off(NO) = 790 M−1 s−1]. Eyring analyses of the NO-independent pathways for these two proteins revealed a significantly larger (ca. 27 cal mol−1 K−1) activation entropy (ΔS‡) in the cross-link-deficient mutant. Our results suggest that the Lys–heme cross-link confers rigidity to the positioning of the heme P460 cofactor to avoid the fast NO-dependent His dissociation pathway and subsequent formation of the off-pathway 5c {FeNO}7 species. The relevance of these findings to NO signaling proteins such as heme-nitric oxide/oxygen binding (H-NOX) is also discussed.
ABSTRACT: Regularization is often utilized to elicit the desired physical results from experimental data. The recent development of a denoising procedure yielding about 2 orders of magnitude in improvement in SNR obviates the need for regularization, which achieves a compromise between canceling effects of noise and obtaining an estimate of the desired physical results. We show how singular value decomposition (SVD) can be employed directly on the denoised data, using pulse dipolar electron spin resonance experiments as an example. Such experiments are useful in measuring distances and their distributions, P(r) between spin labels on proteins. In noise-free model cases exact results are obtained, but even a small amount of noise (e.g., SNR = 850 after denoising) corrupts the solution. We develop criteria that precisely determine an optimum approximate solution, which can readily be automated. This method is applicable to any signal that is currently processed with regularization of its SVD analysis.
ABSTRACT: Nature has long demonstrated the importance of chemical sequence to induce structure and tune physical interactions. Investigating macromolecular structure and dynamics is paramount to understand macromolecular binding and target recognition. To that end, we have synthesized and characterized flexible sulfonated oligothioetheramides (oligoTEAs) by variable temperature pulse field gradient (PFG) NMR, double electron–electron resonance (DEER), and molecular dynamics (MD) simulations to capture their room temperature structure and dynamics in water. We have examined the contributions of synthetic length (2–12mer), pendant group charge, and backbone hydrophobicity. We observe significant entropic collapse, driven in part by backbone hydrophobicity. Analysis of individual monomer contributions revealed larger changes due to the backbone compared to pendant groups. We also observe screening of intramolecular electrostatic repulsions. Finally, we comment on the combination of DEER and PFG NMR measurements via Stokes–Einstein–Sutherland diffusion theory. Overall, this sensitive characterization holds promise to enable de novo development of macromolecular structure and sequence–structure–function relationships with flexible, but biologically functional macromolecules.
ABSTRACT: The mechanism of the recently reported photocontrolled cationic polymerization of vinyl ethers was investigated using a variety of catalysts and chain-transfer agents (CTAs) as well as diverse spectroscopic and electrochemical analytical techniques. Our study revealed a complex activation step characterized by one-electron oxidation of the CTA. This oxidation is followed by mesolytic cleavage of the resulting radical cation species, which leads to the generation of a reactive cation–this species initiates the polymerization of the vinyl ether monomer–and a dithiocarbamate radical that is likely in equilibrium with the corresponding thiuram disulfide dimer. Reversible addition–fragmentation type degenerative chain transfer contributes to the narrow dispersities and control over chain growth observed under these conditions. Finally, the deactivation step is contingent upon the oxidation of the reduced photocatalyst by the dithiocarbamate radical concomitant with the production of a dithiocarbamate anion that caps the polymer chain end. The fine-tuning of the electronic properties and redox potentials of the photocatalyst in both the excited and the ground states is necessary to obtain a photocontrolled system rather than simply a photoinitiated system. The elucidation of the elementary steps of this process will aid the design of new catalytic systems and their real-world applications.
ABSTRACT: Proteins are dynamic entities that populate conformational ensembles, and most functions of proteins depend on their dynamic character. Allostery, in particular, relies on ligand-modulated shifts in these conformational ensembles. Hsp70s are allosteric molecular chaperones with conformational landscapes that involve large rearrangements of their two domains (viz. the nucleotide-binding domain and substrate-binding domain) in response to adenine nucleotides and substrates. However, it remains unclear how the Hsp70 conformational ensemble is populated at each point of the allosteric cycle and how ligands control these populations. We have mapped the conformational species present under different ligand-binding conditions throughout the allosteric cycle of the Escherichia coli Hsp70 DnaK by two complementary methods, ion-mobility mass spectrometry and double electron-electron resonance. Our results obtained under biologically relevant ligand-bound conditions confirm the current picture derived from NMR and crystallographic data of domain docking upon ATP binding and undocking in response to ADP and substrate. Additionally, we find that the helical lid of DnaK is a highly dynamic unit of the structure in all ligand-bound states. Importantly, we demonstrate that DnaK populates a partially docked state in the presence of ATP and substrate and that this state represents an energy minimum on the DnaK allosteric landscape. Because Hsp70s are emerging as potential drug targets for many diseases, fully mapping an allosteric landscape of a molecular chaperone like DnaK will facilitate the development of small molecules that modulate Hsp70 function via allosteric mechanisms.
ABSTRACT: Complexin is a small soluble presynaptic protein that interacts with neuronal SNARE proteins in order to regulate synaptic vesicle exocytosis. While the SNARE-binding central helix of complexin is required for both the inhibition of spontaneous fusion and the facilitation of synchronous fusion, the disordered C-terminal domain (CTD) of complexin is specifically required for its inhibitory function. The CTD of worm complexin binds to membranes via two distinct motifs, one of which undergoes a membrane curvature dependent structural transition that is required for efficient inhibition of neurotransmitter release, but the conformations of the membrane-bound motifs remain poorly characterized. Visualizing these conformations is required to clarify the mechanisms by which complexin membrane interactions regulate its function. Here, we employ optical and magnetic resonance spectroscopy to precisely define the boundaries of the two CTD membrane-binding motifs and to characterize their conformations. We show that the curvature dependent amphipathic helical motif features an irregular element of helical structure, likely a pi-bulge, and that this feature is important for complexin inhibitory function in vivo.
ABSTRACT: We report measurements of the frequency and temperature dependence of ferromagnetic resonance (FMR) for a 15-nm-thick yttrium iron garnet (YIG) film grown by off-axis sputtering. Although the FMR linewidth is narrow at room temperature [corresponding to a damping coefficient α=(9.0±0.2)×10−4], comparable to previous results for high-quality YIG films of similar thickness, the linewidth increases strongly at low temperatures, by a factor of almost 30. This increase cannot be explained as due to two-magnon scattering from defects at the sample interfaces. We point out that the increased low-temperature linewidth can be explained by impurity relaxation mechanisms that were elucidated 50 years ago in bulk YIG samples. High-purity starting materials and careful optimization of growth protocols to avoid nonstoichiometries should therefore be employed for making low-temperature thin-film YIG devices.
ABSTRACT: HAMP domains are dimeric, four-helix bundles that transduce conformational signals in bacterial receptors. Genetic studies of the Escherichia coli serine receptor (Tsr) provide an opportunity to understand HAMP conformational behavior in terms of functional output. To increase its stability, the Tsr HAMP domain was spliced into a poly-HAMP unit from the Pseudomonas aeruginosa Aer2 receptor. Within the chimera, the Tsr HAMP undergoes a thermal melting transition at a temperature much lower than that of the Aer2 HAMP domains. Pulse-dipolar electron spin resonance spectroscopy and site-specific spin-labeling confirm that the Tsr HAMP maintains a four-helix bundle. Pulse-dipolar electron spin resonance spectroscopy was also used to study three well-characterized HAMP mutational phenotypes: those that cause flagella rotation that is counterclockwise (CCW) A and kinase-off; CCW B and also kinase-off; and, clockwise (CW) and kinase-on. Conformational properties of the three HAMP variants support a biphasic model of dynamic bundle stability, but also indicate distinct conformational changes within the helix bundle. Functional kinase-on (CW) and kinase-off (CCW A) states differ by concerted changes in the positions of spin-label sites at the base of the bundle. Opposite shifts in the subunit separation distances of neighboring residues at the C-termini of the α1 and α2 helices are consistent with a helix scissors motion or a gearbox rotational model of HAMP activation. In the drastic kinase-off lesion of CCW B, the α1 helices unfold and the α2 helices form a tight two-helix coiled-coil. The substitution of a critical residue in the Tsr N-terminal linker or control cable reduces conformational heterogeneity at the N-terminus of α1 but does not affect structure at the C-terminus of α2. Overall, the data suggest that transitions from on- to off-states involve decreased motional amplitudes of the Tsr HAMP coupled with helix rotations and movements toward a two-helix packing mode.
ABSTRACT: S-Adenosylmethionine (SAM) has a sulfonium ion with three distinct C-S bonds. Conventional radical SAM enzymes use a [4Fe-4S] cluster to cleave homolytically the C5′,adenosine-S bond of SAM to generate a 5′-deoxyadenosyl radical, which catalyzes various downstream chemical reactions. Radical SAM enzymes involved in diphthamide biosynthesis, such as Pyrococcus horikoshii Dph2 (PhDph2) and yeast Dph1-Dph2 instead cleave the Cγ,Met-S bond of methionine to generate a 3-amino-3-carboxylpropyl radical. We here show radical SAM enzymes can be tuned to cleave the third C-S bond to the sulfonium sulfur by changing the structure of SAM. With a decarboxyl SAM analogue (dc-SAM), PhDph2 cleaves the Cmethyl-S bond, forming 5′-deoxy-5′-(3-aminopropylthio) adenosine (dAPTA, 1). The methyl cleavage activity, like the cleavage of the other two C-S bonds, is dependent on the presence of a [4Fe-4S]+ cluster. Electron-nuclear double resonance and mass spectroscopy data suggests that mechanistically one of the S atoms in the [4Fe-4S] cluster captures the methyl group from dc-SAM, forming a distinct EPR-active intermediate, which can transfer the methyl group to nucleophiles such as dithiothreitol. This reveals the [4Fe-4S] cluster in a radical SAM enzyme can be tuned to cleave any one of the three bonds to the sulfonium sulfur of SAM or analogues, and is the first demonstration a radical SAM enzyme could switch from an Fe-based one electron transfer reaction to a S-based two electron transfer reaction in a substrate-dependent manner. This study provides an illustration of the versatile reactivity of Fe-S clusters.
ABSTRACT: The self-assembly of the microtubule associated tau protein into fibrillar cell inclusions is linked to a number of devastating neurodegenerative disorders collectively known as tauopathies. The mechanism by which tau self-assembles into pathological entities is a matter of much debate, largely due to the lack of direct experimental insights into the earliest stages of aggregation. We present pulsed double electron-electron resonance measurements of two key fibril-forming regions of tau, PHF6 and PHF6*, in transient as aggregation happens. By monitoring the end-to-end distance distribution of these segments as a function of aggregation time, we show that the PHF6(*) regions dramatically extend to distances commensurate with extended β-strand structures within the earliest stages of aggregation, well before fibril formation. Combined with simulations, our experiments show that the extended β-strand conformational state of PHF6(*) is readily populated under aggregating conditions, constituting a defining signature of aggregation-prone tau, and as such, a possible target for therapeutic interventions.
ABSTRACT: We adapt a new wavelet-transform-based method of denoising experimental signals to pulse-dipolar electron-spin resonance spectroscopy (PDS). We show that signal averaging times of the time-domain signals can be reduced by as much as 2 orders of magnitude, while retaining the fidelity of the underlying signals, in comparison with noiseless reference signals. We have achieved excellent signal recovery when the initial noisy signal has an SNR ≳ 3. This approach is robust and is expected to be applicable to other time-domain spectroscopies. In PDS, these time-domain signals representing the dipolar interaction between two electron spin labels are converted into their distance distribution functions P(r), usually by regularization methods such as Tikhonov regularization. The significant improvements achieved by using denoised signals for this regularization are described. We show that they yield P(r)'s with more accurate detail and yield clearer separations of respective distances, which is especially important when the P(r)'s are complex. Also, longer distance P(r)'s, requiring longer dipolar evolution times, become accessible after denoising. In comparison to standard wavelet denoising approaches, it is clearly shown that the new method (WavPDS) is superior.
ABSTRACT: The conserved transmembrane protein, HAP2/GCS1, has been linked to fertility in a wide range of taxa and is hypothesized to be an ancient gamete fusogen. Using template-based structural homology modeling, we now show that the ectodomain of HAP2 orthologs from Tetrahymena thermophila and other species adopt a protein fold remarkably similar to the dengue virus E glycoprotein and related class II viral fusogens. To test the functional significance of this predicted structure, we developed a flow-cytometry-based assay that measures cytosolic exchange across the conjugation junction to rapidly probe the effects of HAP2 mutations in the Tetrahymena system. Using this assay, alterations to a region in and around a predicted "fusion loop" in T. thermophila HAP2 were found to abrogate membrane pore formation in mating cells. Consistent with this, a synthetic peptide corresponding to the HAP2 fusion loop was found to interact directly with model membranes in a variety of biophysical assays. These results raise interesting questions regarding the evolutionary relationships of class II membrane fusogens and harken back to a long-held argument that eukaryotic sex arose as the byproduct of selection for the horizontal transfer of a "selfish" genetic element from cell to cell via membrane fusion.
ABSTRACT: Flagellated bacteria modulate their swimming behavior in response to environmental cues through the CheA/CheY signaling pathway. In addition to responding to external chemicals, bacteria also monitor internal conditions that reflect the availability of oxygen, light, and reducing equivalents, in a process termed "energy taxis." In Escherichia coli, the transmembrane receptor Aer is the primary energy sensor for motility. Genetic and physiological data suggest that Aer monitors the electron transport chain through the redox state of its FAD cofactor. However, direct biochemical data correlating FAD redox chemistry with CheA kinase activity have been lacking. Here, we test this hypothesis via functional reconstitution of Aer into nanodiscs. As purified, Aer contains fully oxidized FAD, which can be chemically reduced to the anionic semiquinone (ASQ). Oxidized Aer activates CheA, whereas ASQ Aer reversibly inhibits CheA. Under these conditions, Aer cannot be further reduced to the hydroquinone, in contrast to the proposed Aer signaling model. Pulse ESR spectroscopy of the ASQ corroborates a potential mechanism for signaling in that the resulting distance between the two flavin-binding PAS (Per-Arnt-Sim) domains implies that they tightly sandwich the signal-transducing HAMP domain in the kinase-off state. Aer appears to follow oligomerization patterns observed for related chemoreceptors, as higher loading of Aer dimers into nanodiscs increases kinase activity. These results provide a new methodological platform to study Aer function along with new mechanistic details into its signal transduction process.
SIGNIFICANCE: Nitrous oxide (N2O) is a potent ozone-depleting greenhouse gas. This work identifies a means by which N2O is generated during nitrification, or biological ammonia oxidation. Fertilizer use in agriculture stimulates nitrification, thus increasing the volume of N2O emissions worldwide. The results presented herein will inform models and strategies toward optimized, sustainable agriculture. Moreover, these results highlight a rare example of biological N–N bond formation.
ABSTRACT: Ammonia oxidizing bacteria (AOB) are major contributors to the emission of nitrous oxide (N2O). It has been proposed that N2O is produced by reduction of NO. Here, we report that the enzyme cytochrome (cyt) P460 from the AOB Nitrosomonas europaea converts hydroxylamine (NH2OH) quantitatively to N2O under anaerobic conditions. Previous literature reported that this enzyme oxidizes NH2OH to nitrite (NO2-) under aerobic conditions. Although we observe NO2- formation under aerobic conditions, its concentration is not stoichiometric with the NH2OH concentration. By contrast, under anaerobic conditions, the enzyme uses 4 oxidizing equivalents (eq) to convert 2 eq of NH2OH to N2O. Enzyme kinetics coupled to UV/visible absorption and electron paramagnetic resonance (EPR) spectroscopies support a mechanism in which an FeIII–NH2OH adduct of cyt P460 is oxidized to an {FeNO}6 unit. This species subsequently undergoes nucleophilic attack by a second equivalent of NH2OH, forming the N–N bond of N2O during a bimolecular, rate-determining step. We propose that NO2- results when nitric oxide (NO) dissociates from the {FeNO}6 intermediate and reacts with dioxygen. Thus, NO2- is not a direct product of cyt P460 activity. We hypothesize that the cyt P460 oxidation of NH2OH contributes to NO and N2O emissions from nitrifying microorganisms.
ABSTRACT: Computer simulations are used to design more hydrated bilayers, formed from amine-modified porphyrin-phospholipids (PoPs). Experiments confirm that the new constructs give rise to bilayers with greater water content. When chelated with manganese, amine-modified PoPs provide improved contrast for magnetic resonance and are safely used for imaging in vivo.
ABSTRACT: The tryptophan 191 cation radical of cytochrome c peroxidase (CcP) compound I (Cpd I) mediates long-range electron transfer (ET) to cytochrome c (Cc). Here we test the effects of chemical substitution at position 191. CcP W191Y forms a stable tyrosyl radical upon reaction with peroxide and produces spectral properties similar to those of Cpd I but has low reactivity toward reduced Cc. CcP W191G and W191F variants also have low activity, as do redox ligands that bind within the W191G cavity. Crystal structures of complexes between Cc and CcP W191X (X = Y, F, or G), as well as W191G with four bound ligands reveal similar 1:1 association modes and heme pocket conformations. The ligands display structural disorder in the pocket and do not hydrogen bond to Asp235, as does Trp191. Well-ordered Tyr191 directs its hydroxyl group toward the porphyrin ring, with no basic residue in the range of interaction. CcP W191X (X = Y, F, or G) variants substituted with zinc-porphyrin (ZnP) undergo photoinduced ET with Cc(III). Their slow charge recombination kinetics that result from loss of the radical center allow resolution of difference spectra for the charge-separated state [ZnP+, Cc(II)]. The change from a phenyl moiety at position 191 in W191F to a water-filled cavity in W191G produces effects on ET rates much weaker than the effects of the change from Trp to Phe. Low net reactivity of W191Y toward Cc(II) derives either from the inability of ZnP+ or the Fe-CcP ferryl to oxidize Tyr or from the low potential of the resulting neutral Tyr radical.
ABSTRACT: Pyrococcus horikoshii Dph2 (PhDph2) is an unusual radical S-adenosylmethionine (SAM) enzyme involved in the first step of diphthamide biosynthesis. It catalyzes the reaction by cleaving SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. To probe the reaction mechanism, we synthesized a SAM analogue (SAMCA), in which the ACP group of SAM is replaced with a 3-carboxyallyl group. SAMCA is cleaved by PhDph2, yielding a paramagnetic (S = 1/2) species, which is assigned to a complex formed between the reaction product, α-sulfinyl-3-butenoic acid, and the [4Fe-4S] cluster. Electron–nuclear double resonance (ENDOR) measurements with 13C and 2H isotopically labeled SAMCA support a π-complex between the C=C double bond of α-sulfinyl-3-butenoic acid and the unique iron of the [4Fe-4S] cluster. This is the first example of a radical SAM-related [4Fe-4S]+ cluster forming an organometallic complex with an alkene, shedding additional light on the mechanism of PhDph2 and expanding our current notions for the reactivity of [4Fe-4S] clusters in radical SAM enzymes.
ABSTRACT: The M2 protein from influenza A plays important roles in its viral cycle. It contains a single transmembrane helix, which oligomerizes into a homotetrameric proton channel that conducts in the low-pH environment of the host-cell endosome and Golgi apparatus, leading to virion uncoating at an early stage of infection. We studied conformational rearrangements that occur in the M2 core transmembrane domain residing on the lipid bilayer, flanked by juxtamembrane residues (M2TMD21–49 fragment), upon its interaction with amantadine drug at pH 5.5 when M2 is conductive. We also tested the role of specific mutation and lipid chain length. Electron spin resonance (ESR) spectroscopy and electron microscopy were applied to M2TMD21–49, labeled at the residue L46C with either nitroxide spin-label or Nanogold® reagent, respectively. Electron microscopy confirmed that M2TMD21–49 reconstituted into DOPC/POPS at 1:10,000 peptide-to-lipid molar ratio (P/L) either with or without amantadine, is an admixture of monomers, dimers, and tetramers, confirming our model based on a dimer intermediate in the assembly of M2TMD21–49. As reported by double electron-electron resonance (DEER), in DOPC/POPS membranes amantadine shifts oligomer equilibrium to favor tetramers, as evidenced by an increase in DEER modulation depth for P/L's ranging from 1:18,000 to 1:160. Furthermore, amantadine binding shortens the inter-spin distances (for nitroxide labels) by 5–8 Å, indicating drug induced channel closure on the C-terminal side. No such effect was observed for the thinner membrane of DLPC/DLPS, emphasizing the role of bilayer thickness. The analysis of continuous wave (cw) ESR spectra of spin-labeled L46C residue provides additional support to a more compact helix bundle in amantadine-bound M2TMD21–49 through increased motional ordering. In contrast to wild-type M2TMD21–49, the amantadine-bound form does not exhibit noticeable conformational changes in the case of G34A mutation found in certain drug-resistant influenza strains. Thus, the inhibited M2TMD21–49 channel is a stable tetramer with a closed C-terminal exit pore. This work is aimed at contributing to the development of structure-based anti-influenza pharmaceuticals.
ABSTRACT: We have shown that de novo designed peptides self-assemble in the presence of copper to create supramolecular assemblies capable of carrying out the oxidation of dimethoxyphenol in the presence of dioxygen. Formation of the supramolecular assembly, which is akin to a protein fold, is critical for productive catalysis since peptides possessing the same functional groups but lacking the ability to self-assemble do not catalyze substrate oxidation. The ease with which we have discovered robust and productive oxygen activation catalysts suggests that these prion-like assemblies might have served as intermediates in the evolution of enzymatic function and opens the path for the development of new catalyst nanomaterials.
ABSTRACT: Restricted motions in proteins (e.g., N–H bond dynamics) are studied effectively with NMR. By analogy with restricted motions in liquid crystals (LC), the local ordering has in the past been primarily represented by potentials comprising the L = 2, |K| = 0, 2 spherical harmonics. However, probes dissolved in LCs experience nonpolar ordering, often referred to as alignment, while protein-anchored probes experience polar ordering, often referred to as orientation. In this study we investigate the role of local (site) symmetry in the context of the polarity of the local ordering. We find that potentials comprising the L = 1, |K| = 0, 1 spherical harmonics represent adequately polar ordering. It is useful to characterize potential symmetry in terms of the irreducible representations of D2h point group, which is already implicit in the definition of the rotational diffusion tensor. Thus, the relevant rhombic L = 1 potentials have B1u and B3u symmetry whereas the relevant rhombic L = 2 potentials have Ag symmetry. A comprehensive scheme where local potentials and corresponding probability density functions (PDFs) are represented in Cartesian and spherical coordinates clarifies how they are affected by polar and nonpolar ordering. The Cartesian coordinates are chosen so that the principal axis of polar axial PDF is pointing along the z-axis, whereas the principal axis of the nonpolar axial PDF is pointing along ±z. Two-term axial potentials with 1 ≤ L ≤ 3 exhibit substantial diversity; they are expected to be useful in NMR-relaxation-data-fitting. It is shown how potential coefficients are reflected in the experimental order parameters. The comprehensive scheme representing local potentials and PDFs is exemplified for the L = 2 case using experimental data from 15N-labeled plexin-B1 and thioredoxin, 2H-, and 13C-labeled benzenehexa-n-alkanoates, and nitroxide-labeled T4 lysozyme. Future prospects for improved ordering analysis based on combined atomistic and mesoscopic approaches are delineated.
ABSTRACT: Detailed EPR investigations on as-grown and annealed TiO2 nanoparticles in the anatase and rutile phases were carried out at X-band (9.6 GHz) at 77, 120–300 K and at 236 GHz at 292 K. The analysis of EPR data for as-grown and annealed anatase and rutile samples revealed the presence of several paramagnetic centers: Ti3+, O−, adsorbed oxygen (O2−) and oxygen vacancies. On the other hand, in as-grown rutile samples, there were observed EPR lines due to adsorbed oxygen (O2−) and the Fe3+ ions in both Ti4+ substitutional positions, with and without coupling to an oxygen vacancy in the near neighborhood. Anatase nanoparticles were completely converted to rutile phase when annealed at 1000° C, exhibiting EPR spectra similar to those exhibited by the as-grown rutile nanoparticles. The high-frequency (236 GHz) EPR data on anatase and rutile samples, recorded in the region about g=2.0 exhibit resolved EPR lines, due to O− and O2− ions enabling determination of their g-values with higher precision, as well as observation of hyperfine sextets due to Mn2+ and Mn4+ ions in anatase.
ABSTRACT: The endosomal sorting complexes required for transport (ESCRTs) constitute hetero-oligomeric machines that catalyze multiple topologically similar membrane-remodeling processes. Although ESCRT-III subunits polymerize into spirals, how individual ESCRT-III subunits are activated and assembled together into a membrane-deforming filament remains unknown. Here, we determine X-ray crystal structures of the most abundant ESCRT-III subunit Snf7 in its active conformation. Using pulsed dipolar electron spin resonance spectroscopy (PDS), we show that Snf7 activation requires a prominent conformational rearrangement to expose protein-membrane and protein-protein interfaces. This promotes the assembly of Snf7 arrays with ˜30 Å periodicity into a membrane-sculpting filament. Using a combination of biochemical and genetic approaches, both in vitro and in vivo, we demonstrate that mutations on these protein interfaces halt Snf7 assembly and block ESCRT function. The architecture of the activated and membrane-bound Snf7 polymer provides crucial insights into the spatially unique ESCRT-III-mediated membrane remodeling.
ABSTRACT: Viral glycoproteins, such as influenza hemagglutinin (HA) and human immunodeficiency virus gp41, are anchored by a single helical segment transmembrane domain (TMD) on the viral envelope membrane. The fusion peptides (FP) of the glycoproteins insert into the host membrane and initiate membrane fusion. Our previous study showed that the FP or TMD alone perturbs membrane structure. Interaction between the influenza HA FP and TMD has previously been shown, but its role is unclear. We used PC spin labels dipalmitoylphospatidyl-tempo-choline (on the headgroup), 5PC and 14PC (5-C and 14-C positions on the acyl chain) to detect the combined effect of FP-TMD interaction by titrating HA FP to TMD-reconstituted 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol)/cholesterol lipid bilayers using electron spin resonance. We found that the FP-TMD increases the lipid order at all positions, which has a greater lipid ordering effect than the sum of the FP or TMD alone, and this effect reaches deeper into the membranes. Although HA-mediated membrane fusion is pH dependent, this combined effect is observed at both pH 5 and pH 7. In addition to increasing lipid order, multiple components are found for 5PC at increased concentration of FP-TMD, indicating that distinct domains are induced. However, the mutation of Gly1 in the FP and L187 in the TMD eliminates the perturbations, consistent with their fusogenic phenotypes. Electron spin resonance on spin-labeled peptides confirms these observations. We suggest that this interaction may provide a driving force in different stages of membrane fusion: initialization, transition from hemifusion stalk to transmembrane contact, and fusion pore formation.
ABSTRACT: Light–oxygen–voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications.
ABSTRACT: Cyclooxygenases (COXs) are heme-containing sequence homodimers that utilize tyrosyl radical-based catalysis to oxygenate substrates. Tyrosyl radicals are formed from a single turnover of substrate in the peroxidase active site generating an oxy-ferryl porphyrin cation radical intermediate that subsequently gives rise to a Tyr-385 radical in the cyclooxygenase active site and a Tyr-504 radical nearby. We have utilized double-quantum coherence (DQC) spectroscopy to determine the distance distributions between Tyr-385 and Tyr-504 radicals in COX-2. The distances obtained with DQC confirm that Tyr-385 and Tyr-504 radicals were generated in each monomer and accurately match the distances measured in COX-2 crystal structures.
ABSTRACT: The large values of spin relaxation enhancement (RE) for PC spin-labels in the phospholipid membrane induced by paramagnetic metal salts dissolved in the aqueous phase can be explained by Heisenberg spin exchange due to conformational fluctuations of the nitroxide group as a result of membrane fluidity, flexibility of lipid chains, and, possibly, amphiphilic nature of the nitroxide label. Whether the magnetic interaction occurs predominantly via Heisenberg spin exchange (Ni) or by the dipole–dipole (Gd) mechanism, it is essential for the paramagnetic ion to get into close proximity to the nitroxide moiety for efficient RE. For different salts of Ni the RE in phosphatidylcholine membranes follows the anionic Hofmeister series and reflects anion adsorption followed by anion-driven attraction of paramagnetic cations on the choline groups. This adsorption is higher for chaotropic ions, e.g., perchlorate. (A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules.) However, there is no anionic dependence of RE for model membranes made from negatively charged lipids devoid of choline groups. We used Ni-induced RE to study the thermodynamics and electrostatics of ion/membrane interactions. We also studied the effect of membrane composition and the phase state on the RE values. In membranes with cholesterol a significant difference is observed between PC labels with nitroxide tethers long enough vs not long enough to reach deep into the membrane hydrophobic core behind the area of fused cholesterol rings. This study indicates one must be cautious in interpreting data obtained by PC labels in fluid membranes in terms of probing membrane properties at different immersion depths when it can be affected by paramagnetic species at the membrane surface.
ABSTRACT: Deuterium line shape analysis from mobile C–D and C–CD3 groups has emerged as a particularly useful tool for studying dynamics in the solid state. The theoretical models devised so far consist typically of sets of independent dynamic modes. Each such mode is simple and usually case-specific. In this scenario, model improvement entails adding yet another mode (thereby changing the overall model), comparison of different cases is difficult, and ambiguity is unavoidable. We recently developed the microscopic order macroscopic disorder (MOMD) approach as a single-mode alternative. In MOMD, the local spatial restrictions are expressed by an anisotropic potential, the local motion by a diffusion tensor, and the local molecular geometry by relative (magnetic and model-related) tensor orientations, all of adjustable symmetry. This approach provides a consistent method of analysis, thus resolving the issues above. In this study, we apply MOMD to PS-adsorbed LKα14 peptide and dimethylammonium tetraphenylborate (C–CD3 and N–CD3 dynamics, respectively), as well as HhaI methyltransferase target DNA and phase III of benzene-6-hexanoate (C–D dynamics). The success with fitting these four disparate cases, as well as the two cases in the previous report, demonstrates the generality of this MOMD-based approach. In this study, C–D and C–CD3 are both found to execute axial diffusion (rates R⊥ and R∥) in the presence of a rhombic potential given by the L = 2 spherical harmonics (coefficients c02 and c22). R⊥ (R∥) is in the 102–103 (104–105) s–1 range, and c02 and c22 are on the order of 2–3 kBT. Specific parameter values are determined for each mobile site. The diffusion and quadrupolar tensors are tilted at either 120° (consistent with trans–gauche isomerization) or nearly 110.5° (consistent with methyl exchange). Future prospects include extension of the MOMD formalism to include MAS, and application to 15N and 13C nuclei.
ABSTRACT: M2 from influenza A virus functions as an oligomeric proton channel essential for the viral cycle, hence it is a high-priority pharmacological target whose structure and functions require better understanding. We studied the mechanism of M2 transmembrane domain (M2TMD) assembly in lipid membranes by the powerful biophysical technique of double electron-electron resonance (DEER) spectroscopy. By varying the M2TMD-to-lipid molar ratio over a wide range from 1:18,800 to 1:160, we found that M2TMD exists as monomers, dimers and tetramers whose relative populations shift to tetramers with the increase of peptide-to-lipid (P/L) molar ratio. Our results strongly support the tandem mechanism of M2 assembly that is monomers-to-dimer then dimers-to-tetramer, since tight dimers are abundant at small P/L’s and thereafter they assemble as dimers of dimers in weaker tetramers. The stepwise mechanism found for a single-pass membrane protein oligomeric assembly should contribute to the knowledge of the association steps in membrane protein folding.
ABSTRACT: Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to form extended membrane arrays that receive and transduce environmental signals. A receptor trimers-of-dimers resides at each vertex of the hexagonal protein lattice. CheA is fully activated and regulated when it is integrated into the receptor assembly. To mimic these states in solution, we have engineered chemoreceptor cytoplasmic kinase-control modules (KCMs) based on the Escherichia coli aspartate receptor Tar that are covalently fused and trimerized by a foldon domain (TarFO). Small-angle X-ray scattering, multi-angle light scattering, and pulsed-dipolar electron spin resonance spectroscopy of spin-labeled proteins indicate that the TarFO modules assemble into homogeneous trimers wherein the protein interaction regions closely associate at the end opposite to the foldon domains. The TarFO variants greatly increase the saturation levels of phosphorylated CheA (CheA-P), indicating that the association with a trimer of receptor dimers changes the fraction of active kinase. However, the rate constants for CheA-P formation with the Tar variants are low compared to those for autophosphorylation by free CheA, and net phosphotransfer from CheA to CheY does not increase commensurately with CheA autophosphorylation. Thus, the Tar variants facilitate slow conversion to an active form of CheA that then undergoes stable autophosphorylation and is capable of subsequent phosphotransfer to CheY. Free CheA is largely incapable of phosphorylation but contains a small active fraction. Addition of TarFO to CheA promotes a planar conformation of the regulatory domains consistent with array models for the assembly state of the ternary complex and different from that observed with a single inhibitory receptor. Introduction of TarFO into E. coli cells activates endogenous CheA to produce increased clockwise flagellar rotation, with the effects increasing in the presence of the chemotaxis methylation system (CheB/CheR). Overall, the TarFO modules demonstrate that trimerized signaling tips self-associate, bind CheA and CheW, and facilitate conversion of CheA to an active conformation.
ABSTRACT: The development, applications, and current challenges of the pulsed ESR technique of two-dimensional Electron-Electron Double Resonance (2D ELDOR) are described. This is a three-pulse technique akin to 2D Exchange Nuclear Magnetic Resonance, but involving electron spins, usually in the form of spin-probes or spin-labels. As a result, it required the extension to much higher frequencies, i.e., microwaves, and much faster time scales, with π/2 pulses in the 2-3 ns range. It has proven very useful for studying molecular dynamics in complex fluids, and spectral results can be explained by fitting theoretical models (also described) that provide a detailed analysis of the molecular dynamics and structure. We discuss concepts that also appear in other forms of 2D spectroscopy but emphasize the unique advantages and difficulties that are intrinsic to ESR. Advantages include the ability to tune the resonance frequency, in order to probe different motional ranges, while challenges include the high ratio of the detection dead time vs. the relaxation times. We review several important 2D ELDOR studies of molecular dynamics. (1) The results from a spin probe dissolved in a liquid crystal are followed throughout the isotropic → nematic → liquid-like smectic → solid-like smectic → crystalline phases as the temperature is reduced and are interpreted in terms of the slowly relaxing local structure model. Here, the labeled molecule is undergoing overall motion in the macroscopically aligned sample, as well as responding to local site fluctuations. (2) Several examples involving model phospholipid membranes are provided, including the dynamic structural characterization of the boundary lipid that coats a transmembrane peptide dimer. Additionally, subtle differences can be elicited for the phospholipid membrane phases: liquid disordered, liquid ordered, and gel, and the subtle effects upon the membrane, of antigen cross-linking of receptors on the surface of plasma membrane, vesicles can be observed. These 2D ELDOR experiments are performed as a function of mixing time, Tm, i.e., the time between the second and third π/2 pulses, which provides a third dimension. In fact, a fourth dimension may be added by varying the ESR frequency/magnetic field combination. Therefore, (3) it is shown how continuous-wave multifrequency ESR studies enable the decomposition of complex dynamics of, e.g., proteins by virtue of their respective time scales. These studies motivate our current efforts that are directed to extend 2D ELDOR to higher frequencies, 95 GHz in particular (from 9 and 17 GHz), in order to enable multi-frequency 2D ELDOR. This required the development of quasi-optical methods for performing the mm-wave experiments, which are summarized. We demonstrate state-of-the-art 95 GHz 2D ELDOR spectroscopy through its ability to resolve the two signals from a spin probe dissolved in both the lipid phase and the coexisting aqueous phase. As current 95 GHz experiments are restricted by limited spectral coverage of the π/2 pulse, as well as the very short T2 relaxation times of the electron spins, we discuss how these limitations are being addressed.
ABSTRACT: We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces.
ABSTRACT: At the base of the bacterial flagella, a cytoplasmic rotor (the C-ring) generates torque and reverses rotation sense in response to stimuli. The bulk of the C-ring forms from many copies of the proteins FliG, FliM, and FliN, which together constitute the switch complex. To help resolve outstanding issues regarding C-ring architecture, we have investigated interactions between FliM and FliG from Thermotoga maritima with X-ray crystallography and pulsed dipolar ESR spectroscopy (PDS). A new crystal structure of an 11-unit FliG:FliM complex produces a large arc with a curvature consistent with the dimensions of the C-ring. Previously determined structures along with this new structure provided a basis to test switch complex assembly models. PDS combined with mutational studies and targeted cross-linking reveal that FliM and FliG interact through their middle domains to form both parallel and antiparallel arrangements in solution. Residue substitutions at predicted interfaces disrupt higher-order complexes that are primarily mediated by contacts between the C-terminal domain of FliG and the middle domain of a neighboring FliG molecule. Spin separations among multi-labeled components fit a self-consistent model that agree well with electron microscopy images of the C-ring. An activated form of the response regulator CheY destabilizes the parallel arrangement of FliM molecules to perturb FliG alignment in a process that may reflect the onset of rotation switching. These data suggest a model of C-ring assembly in which intermolecular contacts among FliG domains provide a template for FliM assembly and cooperative transitions.
ABSTRACT: Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3′- and 5′-termini of the 27-base mini TAR DNA relocated to opposite ends of a 27 bp RNA-DNA duplex with 76.5 Å between labels, a distance which was consistent with the distance between the two labels in a thermally annealed 27-bp TAR DNA-TAR RNA duplex. Different sets of double labels initially located 26–27 Å apart in the mini TAR DNA upper stem, appropriately altered their interlabel distance to ˜35 Å when a 27 bp TAR DNA-TAR RNA duplex formed, where the formation was caused either through NCp7-induced annealing or by thermal annealing. In summary, clear structural evidence was obtained for the fraying and destabilization brought on by NCp7 in its biochemical function as an annealing agent and for the detailed structural change from stem-loop to duplex RNA-DNA when complementary RNA was present.
SIGNIFICANCE: Bacterial chemoreceptors are a key system for understanding how conformational signals propagate over large distances in transmembrane signaling. We have applied pulsed dipolar ESR spectroscopy of spin-labeled receptors to correlate conformation and dynamics with activity state. We find that the receptor cytoplasmic domain behaves as one large dynamically coupled system, in which activation signals destabilize membrane proximal regions but stabilize the most distal protein interaction tip. Inhibitory signals or adaptations of the receptor through chemical modification produce the opposite changes in conformational properties. This reciprocal coupling of conformational stability provides a versatile mechanism for sending signals throughout large modular proteins.
ABSTRACT: Dynamics are hypothesized to play an important role in the transmission of signals across membranes by receptors. Bacterial chemoreceptors are long helical proteins that consist of a periplasmic ligand-binding domain; a transmembrane region; a cytoplasmic HAMP (histidine kinase, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) domain; and a kinase-control module (KCM). The KCM is further composed of adaptation, hinge, and protein interaction regions (PIRs), the latter of which binds the histidine kinase CheA and adaptor CheW. Fusions of the Escherichia coli aspartate receptor KCM to HAMP domains of defined structure (H1-Tar vs. H1-2-Tar) give opposite responses in phosphotransfer and cellular assays, despite similar binding to CheA and CheW. Pulsed dipolar ESR spectroscopy (PDS) of these isolated on and off dimeric effectors reveals that, in the kinase-on state, the HAMP is more conformationally destabilized compared with the PIR, whereas in the kinase-off state, the HAMP is more compact, and the PIR samples a greater breadth of conformations. On and off HAMP states produce different conformational effects at the KCM junction, but these differences decrease through the adaptation region and into the hinge only to return with the inverted relationship in the PIR. Continuous wave–ESR of the spin-labeled proteins confirms that broader PDS distance distributions correlate with increased rates of dynamics. Conformational breadth in the adaptation region changes with charge alterations caused by modification enzymes. Activating modifications broaden the HAMP conformational ensemble but correspondingly, compact the PIR. Thus, chemoreceptors behave as coupled units, in which dynamics in regions proximal and distal to the membrane change coherently but with opposite sign.
ABSTRACT: Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here we report direct observations of these 'elevator-like' transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the 'humanizing' mutations favour structurally 'unlocked' intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.
ABSTRACT: Deuterium line shape analysis of CD3 groups has emerged as a particularly useful tool for studying microsecond–millisecond protein motions in the solid state. The models devised so far consist of several independently conceived simple jump-type motions. They are comprised of physical quantities encoded in their simplest form; improvements are only possible by adding yet another simple motion, thereby changing the model. The various treatments developed are case-specific; hence comparison among the different systems is not possible. Here we develop a new methodology for 2H NMR line shape analysis free of these limitations. It is based on the microscopic-order-macroscopic-disorder (MOMD) approach. In MOMD motions are described by diffusion tensors, spatial restrictions by potentials/ordering tensors, and geometric features by relative tensor orientations. Jump-type motions are recovered in the limit of large orientational potentials. Model improvement is accomplished by monitoring the magnitude, symmetry, and orientation of the various tensors. The generality of MOMD makes possible comparison among different scenarios. CD3 line shapes from the Chicken Villin Headpiece Subdomain and the Streptomyces Subtilisin Inhibitor are used as experimental examples. All of these spectra are reproduced by using rhombic local potentials constrained for simplicity to be given by the L = 2 spherical harmonics, and by axial diffusion tensors. Potential strength and rhombicity are found to be ca. 2–3 kBT. The diffusion tensor is tilted at 120° from the C–CD3 axis. The perpendicular (parallel) correlation times for local motion are 0.1–1.0 ms (3.3–30 μs). Activation energies in the 1.1–8.0 kcal/mol range are estimated. Future prospects include extension to the 2H relaxation limit, application to the 15N and 13C NMR nuclei, and accounting for collective motions and anisotropic media.
SIGNIFICANCE: Mutations in human Cu, Zn superoxide dismutase (SOD) cause the motor neuron disease ALS. To better understand why, we compared the aggregation, metal binding, and conformational dynamics of normal and mutant SOD proteins by using the biophysical techniques of X-ray scattering, inductively coupled plasma MS, and ESR spectroscopy. For SOD proteins with defects at a mutational hotspot, we found that copper deficiency, flexibility, and aggregation paralleled clinical severity in ALS patients. These data support a unifying protein framework destabilization mechanism for SOD-linked ALS and thereby point to potential therapies for this lethal condition with few treatment options.
ABSTRACT: Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu2+ binding site and defined cross-dimer copper–copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.
ABSTRACT: We demonstrate the ability of pulsed dipolar electron spin resonance (ESR) spectroscopy (PDS) to report on the conformation of Cu-Zn superoxide dismutase (SOD1) through the sensitive measurement of dipolar interactions between inherent Cu2+ ions. Although the extent and the anisotropy of the Cu ESR spectrum provides challenges for PDS, Ku-band (17.3 GHz) double electron-electron resonance and double-quantum coherence variants of PDS coupled with distance reconstruction methods recover Cu-Cu distances in good agreement with crystal structures. Moreover, Cu-PDS measurements expose distinct differences between the conformational properties of wild-type SOD1 and a single-residue variant (I149T) that leads to the disease amyotrophic lateral sclerosis (ALS). The I149T protein displays a broader Cu-Cu distance distribution within the SOD1 dimer compared to wild-type. In a nitroxide (NO)-labeled sample, distance distributions obtained from Cu-Cu, Cu-NO, and NO-NO separations reveal increased structural heterogeneity within the protein and a tendency for mutant dimers to associate. In contrast, perturbations caused by the ALS mutation are completely masked in the crystal structure of I149T. Thus, PDS readily detects alterations in metalloenzyme solution properties not easily deciphered by other methods and in doing so supports the notion that increased range of motion and associations of SOD1 ALS variants contribute to disease progression.
ABSTRACT: Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger α-dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We therefore developed a fluorimetric assay that reliably reports on oligomeric state changes of α with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-α, covalently labeled with two fluorophores, allows for direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of α self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation.
ABSTRACT: Tau is a microtubule-associated protein that is genetically linked to dementia and linked to Alzheimer's disease via its presence in intraneuronal neurofibrillary tangle deposits, where it takes the form of aggregated paired helical and straight filaments. Although the precise mechanisms by which tau contributes to neurodegeneration remain unclear, tau aggregation is commonly considered to be a critical component of tau-mediated pathogenicity. Nevertheless, the context in which tau aggregation begins in vivo is unknown. Tau is enriched in membrane-rich neuronal structures such as axons and growth cones, and can interact with membranes both via intermediary proteins and directly via its microtubule-binding domain (MBD). Membranes efficiently facilitate tau aggregation in vitro, and may therefore provide a physiologically relevant context for nucleating tau aggregation in vivo. Furthermore, tau-membrane interactions may potentially play a role in tau's poorly understood normal physiological functions. Despite the potential importance of direct tau-membrane interactions for tau pathology and physiology, the structural mechanisms that underlie such interactions remain to be elucidated. Here, we employ electron spin resonance spectroscopy to investigate the secondary and long-range structural properties of the MBD of three-repeat tau isoforms when bound to lipid vesicles and membrane mimetics. We show that the membrane interactions of the tau MBD are mediated by short amphipathic helices formed within each of the MBD repeats in the membrane-bound state. To our knowledge, this is the first detailed elucidation of helical tau structure in the context of intact lipid bilayers. We further show, for the first time (to our knowledge), that these individual helical regions behave as independent membrane-binding sites linked by flexible connecting regions. These results represent the first (to our knowledge) detailed structural view of membrane-bound tau and provide insights into potential mechanisms for membrane-mediated tau aggregation. Furthermore, the results may have implications for the structural basis of tau-microtubule interactions and microtubule-mediated tau aggregation.
ABSTRACT: DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Polε from Saccharomyces cerevisiae is composed of four subunits—Pol2, Dpb2, Dpb3, and Dpb4. Here, we report the presence of a [Fe-S] cluster directly within the active polymerase domain of Pol2 (residues 1–1187). We show that binding of the [Fe-S] cluster is mediated by cysteines in an insertion (Pol2ins) that is conserved in Pol2 orthologs but is absent in the polymerase domains of Polα, Polδ, and Polζ. We also show that the [Fe-S] cluster is required for Pol2 polymerase activity but not for its exonuclease activity. Collectively, our work suggests that Polε is perhaps more sensitive than other DNA polymerases to changes in oxidative stress in eukaryotic cells.
ABSTRACT: Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. The biosynthesis of diphthamide was proposed to involve three steps. The first step is the transfer of the 3-amino-3-carboxypropyl group from S-adenosyl-L-methionine (SAM) to the histidine residue of EF2, forming a C–C bond. Previous genetic studies showed this step requires four proteins in eukaryotes, Dph1–Dph4. However, the exact molecular functions for the four proteins are unknown. Previous study showed that Pyrococcus horikoshii Dph2 (PhDph2), a novel iron-sulfur cluster-containing enzyme, forms a homodimer and is sufficient for the first step of diphthamide biosynthesis in vitro. Here we demonstrate by in vitro reconstitution that yeast Dph1 and Dph2 form a complex (Dph1-Dph2) that is equivalent to the homodimer of PhDph2 and is sufficient to catalyze the first step in vitro in the presence of dithionite as the reductant. We further demonstrate that yeast Dph3 (also known as KTI11), a CSL-type zinc finger protein, can bind iron and in the reduced state can serve as an electron donor to reduce the Fe-S cluster in Dph1-Dph2. Our study thus firmly establishes the functions for three of the proteins involved in eukaryotic diphthamide biosynthesis. For most radical SAM enzymes in bacteria, flavodoxins and flavodoxin reductases are believed to serve as electron donors for the Fe-S clusters. The finding that Dph3 is an electron donor for the Fe-S clusters in Dph1-Dph2 is thus interesting and opens up new avenues of research on electron transfer to Fe-S proteins in eukaryotic cells.
ABSTRACT: Fusion between viral envelopes and host cell membranes, which is mediated by special glycoproteins anchored on the viral membrane, is required for HIV viral entry and infection. The HIV gp41 fusion peptide (FP), which initiates membrane fusion, adopts either an α-helical or β-sheeted structure depending on the cholesterol concentration. We used phosphocholine spin labels on the lipid headgroup and different positions on the acyl chain to detect its perturbation on lipid bilayers containing different cholesterol concentrations by electron-spin resonance. Our findings were as follows. 1), gp41 FP affects the lipid order in the same manner as previously shown for influenza hemagglutinin FP, i.e., it has a cooperative effect versus the peptide/lipid ratio, supporting our hypothesis that membrane ordering is a common prerequisite for viral membrane fusion. 2), gp41 FP induces membrane ordering in all lipid compositions studied, whereas a nonfusion mutant FP perturbs lipid order to a significantly smaller extent. 3), In high-cholesterol-containing lipid bilayers, where gp41 FP is in the β-aggregation conformation, its effect on the lipid ordering reaches deeper into the bilayer. The different extent to which the two conformers perturb is correlated with their fusogenicity. The possible role of the two conformers in membrane fusion is discussed.
ABSTRACT: Bacterial receptors typically contain modular architectures with distinct functional domains that combine to send signals in response to stimuli. Although the properties of individual components have been investigated in many contexts, there is little information about how diverse sets of modules work together in full-length receptors. Here, we investigate the architecture of Aer2, a soluble gas-sensing receptor that has emerged as a model for PAS (Per–Arnt–Sim) and poly-HAMP (histidine kinase–adenylyl cyclase–methyl-accepting chemotaxis protein–phosphatase) domain signaling. The crystal structure of the heme-binding PAS domain in the ferric, ligand-free form, in comparison to the previously determined cyanide-bound state, identifies conformational changes induced by ligand binding that are likely essential for the signaling mechanism. Heme-pocket alternations share some similarities with the heme-based PAS sensors FixL and EcDOS but propagate to the Iβ strand in a manner predicted to alter PAS–PAS associations and the downstream HAMP junction within full-length Aer2. Small-angle X-ray scattering of PAS and poly-HAMP domain fragments of increasing complexity allow unambiguous domain assignments and reveal a linear quaternary structure. The Aer2 PAS dimeric crystal structure fits well within ab initio small-angle X-ray scattering molecular envelopes, and pulsed dipolar ESR measurements of inter-PAS distances confirm the crystallographic PAS arrangement within Aer2. Spectroscopic and pull-down assays fail to detect direct interactions between the PAS and HAMP domains. Overall, the Aer2 signaling mechanism differs from the Escherichia coli Aer paradigm, where side-on PAS–HAMP contacts are key. We propose an in-line model for Aer2 signaling, where ligand binding induces alterations in PAS domain structure and subunit association that is relayed through the poly-HAMP junction to downstream domains.
ABSTRACT: Bacterial receptors typically contain modular architectures with distinct functional domains that combine to send signals in response to stimuli. Although the properties of individual components have been investigated in many contexts, there is little information about how diverse sets of modules work together in full-length receptors. Here, we investigate the architecture of Aer2, a soluble gas-sensing receptor that has emerged as a model for PAS (Per–Arnt–Sim) and poly-HAMP (histidine kinase–adenylyl cyclase–methyl-accepting chemotaxis protein–phosphatase) domain signaling. The crystal structure of the heme-binding PAS domain in the ferric, ligand-free form, in comparison to the previously determined cyanide-bound state, identifies conformational changes induced by ligand binding that are likely essential for the signaling mechanism. Heme-pocket alternations share some similarities with the heme-based PAS sensors FixL and EcDOS but propagate to the Iβ strand in a manner predicted to alter PAS–PAS associations and the downstream HAMP junction within full-length Aer2. Small-angle X-ray scattering of PAS and poly-HAMP domain fragments of increasing complexity allow unambiguous domain assignments and reveal a linear quaternary structure. The Aer2 PAS dimeric crystal structure fits well within ab initio small-angle X-ray scattering molecular envelopes, and pulsed dipolar ESR measurements of inter-PAS distances confirm the crystallographic PAS arrangement within Aer2. Spectroscopic and pull-down assays fail to detect direct interactions between the PAS and HAMP domains. Overall, the Aer2 signaling mechanism differs from the Escherichia coli Aer paradigm, where side-on PAS–HAMP contacts are key. We propose an in-line model for Aer2 signaling, where ligand binding induces alterations in PAS domain structure and subunit association that is relayed through the poly-HAMP junction to downstream domains.
ABSTRACT: HAMP domains are signal relay modules in >26,000 receptors of bacteria, eukaryotes, and archaea that mediate processes involved in chemotaxis, pathogenesis, and biofilm formation. We identify two HAMP conformations distinguished by a four- to two-helix packing transition at the C-termini that send opposing signals in bacterial chemoreceptors. Crystal structures of signal-locked mutants establish the observed structure-to-function relationships. Pulsed dipolar electron spin resonance spectroscopy of spin-labeled soluble receptors active in cells verify that the crystallographically defined HAMP conformers are maintained in the receptors and influence the structure and activity of downstream domains accordingly. Mutation of HR2, a key residue for setting the HAMP conformation and generating an inhibitory signal, shifts HAMP structure and receptor output to an activating state. Another HR2 variant displays an inverted response with respect to ligand and demonstrates the fine energetic balance between "on" and "off" conformers. A DExG motif found in membrane proximal HAMP domains is shown to be critical for responses to extracellular ligand. Our findings directly correlate in vivo signaling with HAMP structure, stability, and dynamics to establish a comprehensive model for HAMP-mediated signal relay that consolidates existing views on how conformational signals propagate in receptors. Moreover, we have developed a rational means to manipulate HAMP structure and function that may prove useful in the engineering of bacterial taxis responses.
ABSTRACT: Sodium and aspartate symporter from Pyrococcus horikoshii, GltPh, is a homolog of the mammalian glutamate transporters, homotrimeric integral membrane proteins that control neurotransmitter levels in brain synapses. These transporters function by alternating between outward-facing and inward-facing states, in which the substrate binding site is oriented toward the extracellular space and the cytoplasm, respectively. Here we used double electron-electron resonance (DEER) spectroscopy to probe the structure and the state distribution of the subunits in the trimer in distinct hydrophobic environments of detergent micelles and lipid bilayers. Our experiments reveal a conformational ensemble of protomers that sample the outward-facing and inward-facing states with nearly equal probabilities, indicative of comparable energies, and independently of each other. On average, the distributions varied only modestly in detergent and in bilayers, but in several mutants unique conformations were stabilized by the latter.
ABSTRACT: In recent years electron spin resonance (ESR) has provided the means to obtain structural constraints in the field of structural biology on the nanoscale by measuring distances between paramagnetic species, which usually have been nitroxide spin-labels. These ESR methods enable the measurement of distances over the wide range from ca. 6–10 Å to nearly 90 Å. While cw methods may be used for the shortest distances, it is the pulse methods that enable this wide range, as well as determination of the distributions in distance. In this chapter we first describe the underlying theoretical concepts for understanding the principal pulse methods of double quantum coherence (DQC)-ESR and double-electron–electron-resonance (DEER), which we collectively refer to as Pulse-Dipolar ESR Spectroscopies (PDS). We then provide technical aspects of pulse ESR spectrometers required for high quality PDS studies. This is followed by an extensive description of sensitivity considerations in PDS, based largely upon our highly sensitive 17.3 GHz pulse spectrometer at ACERT. This description also includes a comparison of the effectiveness of the respective PDS pulse methods. In addition, the newer methods of 5-pulse DEER, which enables longer distances to be measured than by standard DEER, and 2D-DQC, which provides a convenient mapping for studying orientational coherence between spin labels and their interspin vector, are described.
ABSTRACT: We describe significantly improved long-distance measurements in biomolecules by use of the new multipulse double electron–electron spin resonance (DEER) illustrated with the example of a five-pulse DEER sequence. In this sequence, an extra pulse at the pump frequency is used compared with standard four-pulse DEER. The position of the extra pulse is fixed relative to the three pulses of the detection sequence. This significantly reduces the effect of nuclear spin-diffusion on the electron-spin phase relaxation, thereby enabling longer dipolar evolution times that are required to measure longer distances. Using spin-labeled T4 lysozyme at a concentration less than 50 μM, as an example, we show that the evolution time increases by a factor of 1.8 in protonated solution and 1.4 in deuterated solution to 8 and 12 μs, respectively, with the potential to increase them further. This enables a significant increase in the measurable distances, improved distance resolution, or both.
ABSTRACT: Lipoxygenase enzymes initiate diverse signaling pathways by specifically directing oxygen to different carbons of arachidonate and other polyunsaturated acyl chains, but structural origins of this specificity have remained unclear. We therefore determined the nature of the lipoxygenase interaction with the polar-end of a paramagnetic lipid by electron paramagnetic resonance spectroscopy. Distances between selected grid points on soybean seed lipoxygenase-1 (SBL1) and a lysolecithin spin-labeled on choline were measured by pulsed (electron) dipolar spectroscopy. The protein grid was designed by structure-based modeling so that five natural side chains were replaced with spin labels. Pairwise distances in 10 doubly spin-labeled mutants were examined by pulsed dipolar spectroscopy, and a fit to the model was optimized. Finally, experimental distances between the lysolecithin spin and each single spin site on SBL1 were also obtained. With these 15 distances, distance geometry localized the polar-end and the spin of the lysolecithin to the region between the two domains in the SBL1 structure, nearest to E236, K260, Q264, and Q544. Mutation of a nearby residue, E256A, relieved the high pH requirement for enzyme activity of SBL1 and allowed lipid binding at pH 7.2. This general approach could be used to locate other flexible molecules in macromolecular complexes.
ABSTRACT: Electron paramagnetic resonance (EPR) at 236.6 and 9.5 GHz probed the tumbling of nitroxide spin probes in the lower stem, in the upper loop, and near the bulge of mini c TAR DNA. High-frequency 236.6 GHz EPR, not previously applied to spin-labeled oligonucleotides, was notably sensitive to fast, anisotropic, hindered local rotational motion of the spin probe, occurring approximately about the NO nitroxide axis. Labels attached to the 2′-aminocytidine sugar in the mini c TAR DNA showed such anisotropic motion, which was faster in the lower stem, a region previously thought to be partially melted. More flexible labels attached to phosphorothioates at the end of the lower stem tumbled isotropically in mini c TAR DNA, mini TAR RNA, and ψ3 RNA, but at 5 °C, the motion became more anisotropic for the labeled RNAs, implying more order within the RNA lower stems. As observed by 9.5 GHz EPR, the slowing of nanosecond motions of large segments of the oligonucleotide was enhanced by increasing the ratio of the nucleocapsid protein NCp7 to mini c TAR DNA from 0 to 2. The slowing was most significant at labels in the loop and near the bulge. At a 4:1 ratio of NCp7 to mini c TAR DNA, all labels reported tumbling times of >5 ns, indicating a condensation of NCp7 and TAR DNA. At the 4:1 ratio, pulse dipolar EPR spectroscopy of bilabels attached near the 3′ and 5′ termini showed evidence of an NCp7-induced increase in the 3′–5′ end-to-end distance distribution and a partially melted stem.
INTRODUCTION: Lipid spin labels containing nitroxide groups at different positions in the fatty acid chain, such as 1-palmitoyl-2-stearoyl-(n-doxyl)-sn-glycero-3-phosphocholines (n-PC spin labels) are a useful and proven tool in lipid research. They have provided important insights into the structure of model and biological membranes, reported on the membrane fluidity, polarity, phase state and presence of microscopic domains, accessibility of different depth positions in the lipid bilayer for oxygen and other polar and non-polar paramagnetic compounds and protein/lipid interactions.
It is generally accepted, that, unlike bulky fluorescent labels, nitroxides are well incorporated into fluid lipid bilayers and not excluded from them. However, it has been shown by NMR that although the most probable location of the nitroxide group for 5-, 10- and 16- PC spin labels in the fluid POPC membrane corresponds to the fully extended conformation, the distribution is relatively broad and other conformations should also be present. Bent conformations were previously found for doxylstearic acids in monomolecular films, water/hydrocarbon emulsion particles and micellar systems. In fluid membranes the fluidity, polarity and accessibility parameters reported by ESR using PC spin labels and n-doxylstearic acids are, in general, change monotonically with an increase in n, although there are indications that the spin label groups on the stearates are located nearer to the membrane exterior than the analogous positions of the unlabeled phospholipid chains. However, in the gel phase, which is characterized by denser chain packing and higher order, the preferential location may be different.
In this chapter we focus on the behavior of PC spin labels in the gel phase and frozen membranes. We show how the superior g-factor resolution of HF ESR provides new insights in this behavior and a new look at the vast body of experimental data accumulated with PC spin labels in the last 30 years. In particular, we revisit so-called "polarity profiles" determined from the g-factor values and hyperfine splittings of PC spin labels in frozen phospholipid membranes with or without cholesterol and show that these values are affected by a number of factors in the membrane composition, chain packing in the lipid phase and folding properties of the sn-2 spin labeled chain PC labels rather than reflect gradients of polarity or water content present in the membrane.
ABSTRACT: Pulsed electron spin resonance (ESR) dipolar spectroscopy (PDS) in combination with site-directed spin labeling is unique in providing nanometer-range distances and distributions in biological systems. To date, most of the pulsed ESR techniques require frozen solutions at cryogenic temperatures to reduce the rapid electron spin relaxation rate and to prevent averaging of electron–electron dipolar interaction due to the rapid molecular tumbling. To enable measurements in liquid solution, we are exploring a triarylmethyl (TAM)-based spin label with a relatively long relaxation time where the protein is immobilized by attachment to a solid support. In this preliminary study, TAM radicals were attached via disulfide linkages to substituted cysteine residues at positions 65 and 80 or 65 and 76 in T4 lysozyme immobilized on Sepharose. Interspin distances determined using double quantum coherence (DQC) in solution are close to those expected from models, and the narrow distance distribution in each case indicates that the TAM-based spin label is relatively localized.
ABSTRACT: Electron transfer or quantum tunneling dynamics for excess or solvated electrons in dilute lithium–ammonia solutions have been studied by pulse electron paramagnetic resonance (EPR) spectroscopy at both X- (9.7 GHz) and W-band (94 GHz) frequencies. The electron spin–lattice (T1) and spin–spin (T2) relaxation data indicate an extremely fast transfer or quantum tunneling rate of the solvated electron in these solutions which serves to modulate the hyperfine (Fermi-contact) interaction with nitrogen nuclei in the solvation shells of ammonia molecules surrounding the localized, solvated electron. The donor and acceptor states of the solvated electron in these solutions are the initial and final electron solvation sites found before, and after, the transfer or tunneling process. To interpret and model our electron spin relaxation data from the two observation EPR frequencies requires a consideration of a multiexponential correlation function. The electron transfer or tunneling process that we monitor through the correlation time of the nitrogen Fermi-contact interaction has a time scale of (1–10) × 10–12 s over a temperature range 230–290 K in our most dilute solution of lithium in ammonia. Two types of electron–solvent interaction mechanisms are proposed to account for our experimental findings. The dominant electron spin relaxation mechanism results from an electron tunneling process characterized by a variable donor–acceptor distance or range (consistent with such a rapidly fluctuating liquid structure) in which the solvent shell that ultimately accepts the transferring electron is formed from random, thermal fluctuations of the liquid structure in, and around, a natural hole or Bjerrum-like defect vacancy in the liquid. Following transfer and capture of the tunneling electron, further solvent-cage relaxation with a time scale of ˜10–13 s results in a minor contribution to the electron spin relaxation times. This investigation illustrates the great potential of multifrequency EPR measurements to interrogate the microscopic nature and dynamics of ultrafast electron transfer or quantum-tunneling processes in liquids. Our results also impact on the universal issue of the role of a host solvent (or host matrix, e.g. a semiconductor) in mediating long-range electron transfer processes and we discuss the implications of our results with a range of other materials and systems exhibiting the phenomenon of electron transfer.
ABSTRACT: Biologically important protein complexes often involve molecular interactions that are low affinity or transient. We apply pulsed dipolar electron spin resonance spectroscopy and site-directed spin labeling in what to our knowledge is a new approach to study aggregation and to identify regions on protein surfaces that participate in weak, but specific molecular interactions. As a test case, we have probed the self-association of the chemotaxis kinase CheA, which forms signaling clusters with chemoreceptors and the coupling protein CheW at the poles of bacterial cells. By measuring the intermolecular dipolar interactions sensed by spin-labels distributed over the protein surface, we show that the soluble CheA kinase aggregates to a small extent through interactions mediated by its regulatory (P5) domain. Direct dipolar distance measurements confirm that a hydrophobic surface at the periphery of P5 subdomain 2 associates CheA dimers in solution. This result is further supported by differential disulfide cross-linking from engineered cysteine reporter sites. We suggest that the periphery of P5 is an interaction site on CheA for other similar hydrophobic surfaces and plays an important role in structuring the signaling particle.
ABSTRACT: An analysis of electron spin resonance (ESR) spectra from compositions along the liquid-ordered (Lo) and liquid-disordered (Ld) coexistence curve from the brain-sphingomyelin/dioleoylphosphatidylcholine/cholesterol (SPM/DOPC/Chol) model lipid system was performed to characterize the dynamic structure on a molecular level of these coexisting phases. We obtained 200 continuous-wave ESR spectra from glycerophospholipid spin-labels labeled at the 5, 7, 10, 12, 14, and 16 carbon positions of the 2nd acyl chain, a sphingomyelin spin-label labeled at the 14 carbon position of the amide-linked acyl chain, a headgroup-labeled glycerophospholipid, a headgroup-labeled sphingomyelin, and the cholesterol analogue spin-label cholestane all within multi-lamellar vesicle suspensions at room temperature. The spectra were analyzed using the MOMD (microscopic-order macroscopic-disorder) model to provide the rotational diffusion rates and order parameters which characterize the local molecular dynamics in these phases. The analysis also incorporated the known critical point and invariant points of the neighboring three-phase triangle along the coexistence curve. The variation in the molecular dynamic structures of coexisting Lo and Ld compositions as one moves toward the critical point is discussed. Based on these results, a molecular model of the Lo phase is proposed incorporating the "condensing effect" of cholesterol on the phospholipid acyl chain dynamics and ordering and the "umbrella model" of the phospholipid headgroup dynamics and ordering.
ABSTRACT: The ESR parameters of PC spin labels in frozen membranes do not simply represent the membrane polarity or water penetration profile. Instead, they show a distribution between hydrogen-bonded (HB) and non-hydrogen-bonded (non-HB) states, which is affected by a number of factors in the membrane composition. Similar to the exclusion of solutes from crystallizing solvents, the pure bulk gel phase excludes nitroxides, forcing acyl chains to take bent conformations. In these conformations, the nitroxide is hydrogen-bonded. Furthermore, upon gradual cooling in the supercooled gel, PC labels undergo slow lateral aggregation, resulting in a broad background signal. However, if the sample is instantly frozen, this background is replaced by the HB component. In membranes with cholesterol, the observed HB/non-HB ratio can best be described by a partition-like equilibrium between nitroxides located in defects of lipid structure within the hydrophobic core and those close to the membrane surface.
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