95 GHz 2D-ELDOR of spin-labeled Peptides in Membranes

2D ELDOR is a pulsed ESR technique that supplies greatly increased resolution to motional dynamics and local structure of biomolecules, such as membrane lipids and proteins, compared to conventional cw ESR techniques. In a recent study of the effect of the ion-channel forming peptide, gramicidin A, (GA) on model membrane structures, we could clearly distinguish and study the properties of both the boundary lipids that coat GA and the bulk lipids. That study at 17 GHz, however, indicated that higher frequency studies are needed, due to the increased orientational resolution they can provide. This enables better discrimination of the details of the ordering and dynamics. We have now extended the capabilities of our new high-power pulsed 95 GHz spectrometer to enable the study of spin-labeled lipids, such as the cholesterol analogue, CSL, and spin-labeled GA (GASL). The great challenges at 95 GHz are to have a sufficiently wide spectral coverage (as much as 350 MHz) to obtain the full nitroxide spin-label spectrum, and to have sufficiently short dead-times in view of the shortened T₂ relaxation times at higher frequencies. We have succeeded, in particular, with macroscopically aligned membrane samples. Below are typical spectra obtained from GASL in aligned DPPC membranes at 7°C. The example shown is for the membrane normal parallel to the dc magnetic field. The parallel orientation shows the predominance of the molecular z-orientation, consistent with the way the GA aligns in DPPC. When the membrane is tilted perpendicular to the dc magnetic field, spectral contributions from the molecular x and y orientations become more pronounced as expected for such an aligned sample. What is of particular interest is that the 2D-ELDOR spectral patterns change with mixing time, T as seen below in figures (a) and (b). This is a clear indication that these 2D-ELDOR spectra are showing slow-motional effects. That is, the various dynamic spin packet contributions to the spectrum are relaxing at different rates, leading to significant spectral changes with T_m. These results are expected to provide insight into the detailed dynamics of GA in the membranes.