Characterization of a Bifunctional Spin Label for the Structure and Dynamics of a Membrane Protein using CW-EPR Spectroscopy

Abstract

Electron Paramagnetic Resonance (EPR) spectroscopy is a powerful structural biology tool when combined with site-directed spin labeling (SDSL) to study the structure and dynamics of peptides and proteins. The most widely used spin label for SDSL is methanthiosulfonate (MTSL), however the flexibility of this spin label introduces greater uncertainties in the EPR measurements obtained for determining structure, side chain dynamics, and backbone motion of membrane protein systems. Most recently, a new bifunctional spin label (BSL) 3,4-Bis-(methanethiosulfonylmethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxy, has been introduced to overcome some of the limitations associated with the MTSL spin label and has been invaluable in determining protein dynamics and inter-residue distances due to its restricted internal motion and fewer size restrictions. While BSL has been successful in providing information about the structure and dynamics of several proteins, the details of its characterization is lacking. The goal of this research is to gain insights into the behavior of the BSL that has been so useful in providing details to characterize a variety of protein systems. Fmoc solid phase peptide synthesis (SPPS) has been used to generate a series of double cysteine mutants of the 23 amino acid α-helical membrane peptide, AChR M2delta at i and i+4 residue positions. BSLs bearing peptides were incorporated into 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) bicelles and multilamellar vesicles. The spectra were collected at different temperatures utilizing continuous wave (CW)-EPR spectroscopy to examine the dynamics of the BSL within a membrane. The EPR spectral lineshape analysis will be conducted to obtain the static and dynamic EPR parameters. This study will provide more accurate insights into the behavior of BSL in order to provide the optimal conditions for other studies that may utilize this promising spin label.