Title: EPR Spectra of Spin-Labeled OmpA Reflect Both Protein Dynamics and Label Configuration in Membrane Mimetic Systems
Abstract: Electron paramagnetic resonance (EPR) spectroscopy coupled with site-directed spin labeling (SDSL) is a powerful tool that can provide information on the structure and dynamics of proteins. It has been used successfully in both water soluble and membrane proteins. Because SDSL has no molecular weight limitation, it can be performed under physiological conditions and can give data on systems inaccessible to techniques such as solution NMR and X-ray crystallography. The EPR spectra of labeled sites in the outer membrane protein OmpA are highly dependent upon environment, and generally the spectra indicate that labels (R1 side chain) have less motional averaging in the presence of lipids than in detergent systems. In proteins, EPR spectra are known to be modulated by backbone dynamics. This suggests that OmpA is more dynamic on the ns timescale in detergents than in lipid bilayers. In the present study, continuous wave (cw) and pulse EPR techniques were used to examine the basis for changes in the EPR lineshapes of spin-labeled OmpA. In aqueous exposed regions of the protein, lineshape changes as a function of environment likely reflect changes in protein dynamics; however, within the membrane hydrocarbon region, changes in label dynamics may be influenced by the configuration of the label. The results suggest that as the polarity and/or proticity of the environment decreases, the R1 side chain preferentially interacts with the protein backbone. This result is supported by double electron-electron resonance (DEER) measurements, which can only be accounted for by changes in label rotameric states as a function of environment. The results indicate that in addition to protein dynamics, EPR spectra may be strongly modulated by spin label interactions with the surface of membrane proteins. (Supported by NIGMS GM-035215.)