Probing the Secondary Structure of Membrane Protein using Bacterial Expression System and Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy

Abstract

Membrane proteins possess a variety of functions essential to the survival of organisms. However, due to the inherent hydrophobic property, it is extremely difficult to gain the structural information. Previously, we probed the structure of an α-helical component of a small model membrane peptide, AchR M2δ, by detecting 2H modulation from a 2H-labeled Val side chain that was 3 or 4 residues away from a nitroxide spin label using ESEEM spectroscopy. In this study, for the first time, ESEEM technique was applied to determine the local secondary structure of a biological membrane protein system using bacterial expression method for incorporating the 2H-labeled Val side chain both in micelle and lipid bilayer environment. An integral membrane protein, KCNE1 with known 3D structure, was utilized as a model membrane protein to test the feasibility and validity of this ESSEM technique. The side chain of valine at position 21 (outside domain), 50 (transmembrane domain), and 95 (cytoplasmic domain) was 2H labeled (denoted as ith position). An MTSL nitroxide spin label was positioned at 2, 3, and 4 residues away (denoted i+2 to i+4, respectively) from 2H-labeled Val residue of each domain. Our results indicated that i+3 and i+4 positions have a significant 2H modulation while 2H modulation is absent at i+2 position, which is consistent with the 3.6 residue/turn periodicity of the α-helix. These results are also consistent with the micelle structure of KCNE1. This ESEEM technique was further validated using the protein incorporated into lipid bilayer of DMPC/DPC bicelle and the transmembrane domain was probed to be α-helical as well. This ESEEM technique is very powerful, efficient, and quick to examine the local secondary structure of any protein system without size limitation.