Utilizing Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy to Probe the Structure of Membrane Proteins

Abstract

New approaches are needed to more efficiently probe the structural properties of membrane proteins. A new approach has been developed to probe the structural properties of membrane peptides and proteins using the pulsed Electron Paramagnetic Resonance (EPR) technique of Electron Spin Echo Envelope Modulation (ESEEM). This technique can measure short-range distances between a nitroxide spin label and a 2H nucleus out to approximately 8Å. For this study a model membrane peptide M2δ, was constructed by solid phase peptide synthesis and inserted into a DMPC/DHPC bicelle membrane. We report for the first time, the direct detection of 2H modulation between a 2H-labeled d8 Val residue and a nitroxide spin label three and four residues away that is characteristic of an alpha-helical secondary structure. Simulations of the ESEEM data reveal a distance of approximately 6.4 +/- 0.5Å that agrees well with molecular modeling studies. ESEEM spectra in this work yielded high-quality data in less than an hour with as little as 35μg of protein sample.