Membrane Protein Secondary Structure Determination via Pulsed EPR ESEEM

Abstract

Membrane proteins are involved in a variety of pertinent roles within the cell such as ion channels, communication, and catalysts for reactions in the membrane. Limited structural information exists for membrane proteins and new approaches need to be developed. A new method has been developed to determine the secondary structure of membrane proteins using pulsed electron paramagnetic resonance (EPR) spectroscopy. The three-pulse electron spin echo envelope modulation (ESEEM) can be used to detect the dipolar coupling between a deuterium-labeled residue and a spin label attached up to 4 residues away (i + 4) and correlate the strength of this coupling with the radial distance between the spin and deuterium labels. In this case, i represents the location of the 2H-labeled residue and 4 represents the location of the spin label 4 residues away. Unique periodic structural difference between an α-helix and a β-sheet can be easily observed in the corresponding ESEEM spectra for i + 2 and i + 3 samples. We have demonstrated these distinct trends with model α-helical (M2δ subunit of the acetylcholine receptor) and β-sheet (first 17-residues of ubiquitin) peptides. Circular Dichorism spectroscopy further confirms the secondary structure of the labeled peptide constructs to ensure the validity of the method. The new ESEEM approach provides site-specific secondary structural information both qualitatively and quantitatively on membrane proteins that otherwise might not have been structurally elucidated.