Aggregation of spin labeled molecules by Dipolar Pulse ESR

Pulse Dipolar ESR (PDS), mainly represented by Double Electron-Electron Resonance (DEER) and Double-Quantum Coherence ESR (DQC) techniques, is now routinely used to measure distances between nitroxide spin-labels introduced into the structure of a variety of biological systems. The methods allow distance measurements over a wide range from about 10 to nearly 80Å. This distance range makes PDS well-suited for studying aggregation and association of peptides and proteins in solutions and lipid membranes. We illustrate this application of PDS with the study of alamethicin aggregation in model lipid membranes. The ESR spectrum of spin-labeled alamethicin in macroscopically-aligned lipid bilayers showed that it is oriented along the bilayer normal with significant exposure of the C-terminus to the water phase. A uniform distribution of spins in the isotropic media yields a simple exponential decay of the time-domain PDS signal, V(t) (see Figure of logV(t) vs. t below). We found that this also holds in multi-bilayer vesicles. This simplifies isolating the dipolar signal of an aggregate (cluster) and determining the asymptotic value. To determine the average size of clusters we detected the dipolar signal for a series of samples with the same content of spin-labeled alamethicin and lipid, but different concentrations of unlabeled alamethicin. The dipolar signal is then isolated from the baseline and normalized by its value at zero time. The normalized intensity is a direct measure of the number of spins, N. It depends on the spectral excitation, p, magnetic dilution, f, and N in the cluster. We found that N ranges from 4 in POPC to 6 in DPPC. Also the size of alamethicin clusters may be obtained from the distance distributions P(r) yielding a larger size of the cluster in DPPC as shown in the figure below, which is consistent with the larger value of N in this lipid.