Probing the Structural Topology of a Membrane Peptide in Mechcanically Aligned Lipid Bilayers using Bifunctional Spin Labeling EPR Spectroscopy

Abstract

Electron Paramagnetic Resonance (EPR) spectroscopy coupled with site-directed spin labeling (SDSL) is a powerful structural biology tool for studying the structural and dynamic properties of peptides, proteins, and nucleic acids. The most commonly used spin label for SDSL is methanthiosufonate (MTSL), however the flexibility of this spin label can introduce greater uncertainties in the EPR measurements for determining structure, side chain orientations and backbone motion of membrane protein systems. Another spin label, 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) has been found to be a useful alternative given its rigid structure, however it is very challenging to introduce this spin label into biological protein systems. The goal of this research is to develop an improved biophysical method for studying the structural and dynamic properties of membrane proteins using EPR spectroscopy that will overcome the limitations associated with MTSL and TOAC. A recently discovered bifunctional spin label (BSL) 3,4-Bis-(methanethiosulfonylmethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxy, will be utilized. Fmoc solid phase peptide synthesis (SPPS) will be used to generate a double cysteine mutant of the 23 amino acid α-helical membrane peptide, AChR M2δ. Once labeled with BSL and incorporated into 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) bilayers, alignment techniques utilizing EPR spectroscopy will be performed to examine the structural topology. This study will provide a structural biology tool that can be used to obtain very accurate and precise EPR measurements to answer several structural and dynamics related questions on membrane protein systems.