HFHF Multifrequency ESR on Aligned Membranes

Aligned membranes incorporating spin labeled peptides are useful models for studying the behavior of biological systems. Exploiting the superior orientational selectivity of high field/high frequency (HF/HF) ESR yields useful insights into the dynamics and structure of these systems. Experiments performed over a range of frequencies allow detailed analysis of the various dynamical modes coupling the peptide to its surroundings. As an example of the superior resolution to structure and dynamics available from HF/HF ESR, we show spectra from spin labeled gramicidin-A’ in DPPC. Above the L_a - P_b transition point, this system forms dimer channels. The spectral signature is a loss of Z-ordering due to tilting of the nitroxide moiety as the channel forms. This conclusion, which could only be reached after extensive spectral analysis at 9GHz, is obvious by inspection of the spectrum at 310K taken at 170GHz shown below. We observe that the spectral weight shifts from the X- and Y- regions to the Z-region upon cooling the sample as shown in the spectrum taken at 283K. The HF/HF spectra were taken on a cw-ESR spectrometer that was built at the Center using the techniques of quasi-optical design and upgraded with recently acquired higher power cw millimeter wave sources. Important information about spin label ordering in aligned membrane samples can also be obtained by varying the sample orientation with respect to the applied magnetic field. Due to larger dielectric losses at higher frequencies, a successful experiment requires thin (< 100 mm) flat samples placed at a node of the millimeter wave E-field. For our fixed resonator geometry, the sample normal is typically parallel to the applied magnetic field, B₀. To obtain high field spectra in an orientation different from this parallel orientation, we use microtome techniques on Iso-Potential Spin-Dry Ultra-centrifugation (ISDU) aligned samples to obtain thin slices of various, controllable, membrane orientations. We have also used this technique in the Center’s 95GHz spectrometer where similar sample constraints affect the performance of the resonator. The sensitivity of the spectra to the sample orientation is evident by inspection.