Nitroxide Spin Label Side Chain Dynamics of Solvent Exposed Sites on Membrane Proteins

Abstract

Understanding the structure and functional dynamics of membrane proteins in their native, hydrophobic environment is key to understanding how proteins function. EPR spectroscopy in combination with site directed spin labeling (SDSL) has the potential to quantify structure and dynamics of proteins of arbitrary weight in their native lipid environment. Several studies have elucidated the structural origins of CW EPR lineshapes of water-soluble proteins; however, CW EPR spectra of nitroxide spin labeled proteins in a detergent/lipid environment have characteristic differences from their water-soluble counterparts. Membrane protein spectra are generally broader and frequently contain multiple spectral components. In this study, membrane exposed sites on model α-helical and β-barrel proteins LeuT and OmpA, respectively, were investigated using X-ray crystallography, mutational analysis, nitroxide side chain derivatives, and spectral simulations in order to obtain a motional model of the nitroxide. For each crystal structure of spin labeled LeuT, the nitroxide ring of the spin label is resolved and makes contacts with hydrophobic residues on the protein surface. In addition to the crystal structures, the spectral effect of nearest neighbor mutations and the spectral simulations suggest the spin label has a greater affinity for its local protein environment compared to water-soluble proteins. These results for spin labeled sites on LeuT and OmpA begin to explain the observed trends in membrane protein CW EPR spectra.