Electron spin resonance in membrane research: protein-lipid interactions from challenging beginnings to state of the art.
Conventional electron paramagnetic resonance (EPR) spectra of lipids that are spin-labelled close to the terminal methyl end of the acyl chains are able to resolve the lipids directly contacting the protein from those in the fluid bilayer regions of the membrane. This allows determination of both the stoichiometry of lipid-protein interaction (i.e., number of lipid sites at the protein perimeter) and the selectivity of the protein for different lipid species (i.e., association constants relative to the background lipid). Spin-label EPR data are summarised for 20 or more different transmembrane peptides and proteins, and 7 distinct species of lipids. Lineshape simulations of the two-component conventional spin-label EPR spectra allow estimation of the rate at which protein-associated lipids exchange with those in the bulk fluid regions of the membrane. For lipids that do not display a selectivity for the protein, the intrinsic off-rates for exchange are in the region of 10 MHz: less than 10x slower than the rates of diffusive exchange in fluid lipid membranes. Lipids with an affinity for the protein, relative to the background lipid, have off-rates for leaving the protein that are correspondingly slower. Non-linear EPR, which depends on saturation of the spectrum at high radiation intensities, is optimally sensitive to dynamics on the timescale of spin-lattice relaxation, i.e., the microsecond regime. Both progressive saturation and saturation transfer EPR experiments provide definitive evidence that lipids at the protein interface are exchanging on this timescale. The sensitivity of non-linear EPR to low frequencies of spin exchange also allows the location of spin-labelled membrane protein residues relative to those of spin-labelled lipids, in double-labelling experiments.
Marsh
Derek
D
Abteilung Spektroskopie, Max-Planck-Institut für biophysikalische Chemie, 37070 Göttingen, Germany. dmarsh@gwdg.de
D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013113:Spin Labels
D008565:Membrane Proteins; D013113:Spin Labels