Nitroxide Spin Label Motion on Helical Membrane Proteins

Understanding the structure and dynamics of membrane proteins in their native, hydrophobic environment is important to understanding how these proteins function. Electron paramagnetic resonance (EPR) spectroscopy in combination with site directed spin labeling (SDSL) can measure dynamics and structure of membrane proteins in their native lipid environment; however, until now the dynamics measured have been qualitative due to limited knowledge of the nitroxide spin labels intramolecular motion in the hydrophobic environment. Although several studies have elucidated the structural origins of EPR lineshapes of water-soluble proteins, EPR spectra of nitroxide spin labeled proteins in detergents or lipids have characteristic differences from their water-soluble counterparts. To elucidate the physical underpinnings of these differences, membrane exposed -helical sites of the leucine transporter, LeuT, from A. aeolicus, were investigated using X-ray crystallography, mutational analysis, nitroxide side chain derivatives, and spectral simulations. For each crystal structure, the nitroxide ring of a disulfide-linked spin label side chain (R1) is resolved and makes contacts with hydrophobic residues on the protein surface. The spin label at site I204 on LeuT makes a non-traditional hydrogen bond with the ortho hydrogen on its nearest neighbor F208, whereas the spin label at site F177 makes multiple van der Waals contacts with a hydrophobic pocket formed with an adjacent helix. These results coupled with the spectral effect of mutating the i ± 3, 4 residues suggest that the spin label has a greater affinity for its local iii protein environment in the low dielectric than on a water-soluble protein surface. The simulations of the EPR spectra presented here suggest the spin label oscillates about the terminal bond nearest the ring while maintaining weak contact with the protein surface. Combined, the results provide a starting point for determining a motional model for R1 on membrane proteins allowing quantification of nitroxide dynamics in the aliphatic environment of detergent and lipids. In addition, initial results towards reliably modeling the R1 conformational space for NMR paramagnetic relaxation enhancement distance determination will be presented. To my advisor, for my growth as a scientist and for all the opportunities I had at UVA. To my wife, for everything.

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