Abstract
A new approach has been developed to probe the structural properties of membrane peptides and proteins using the pulsed electron paramagnetic resonance technique of electron spin echo envelope modulation (ESEEM) spectroscopy and the alpha-helical M2 delta subunit of the acetylcholine receptor incorporated into phospholipid bicelles. To demonstrate the practicality of this method, a cysteine-mutated nitroxide spin label (SL) is positioned 1, 2, 3, and 4 residues away from a fully deuterated Val side chain (denoted i + 1 to i + 4). The characteristic periodicity of the alpha-helical structure gives rise to a unique pattern in the ESEEM spectra. In the i + 1 and i + 2 samples, the H-2 nuclei are too far away to be detected. However, with the 3.6 residue per turn pattern of an alpha-helix, the i + 3 and i + 4 samples reveal a strong signal from the H-2 nuclei of the Val side chain. Modeling studies verify these data suggesting that the closest H-2-labeled Val to SL distance would in fact be expected in the i + 3 and i + 4 samples. This technique is very advantageous, because it provides pertinent qualitative structural information on an inherently difficult system like membrane proteins in a short period of time (minutes) with small amounts of protein (mu g).