Lipid and membrane mimetic environments modulate spin label side chain configuration in the outer membrane protein A.
In the present work, the factors that determine EPR line shapes from spin labels at the protein-hydrocarbon interface of a β-barrel membrane protein are examined. The EPR spectra from hydrocarbon facing sites in the outer membrane protein A (OmpA) are highly dependent upon the detergent or lipid into which OmpA is reconstituted. In general, line shapes at these sites are correlated with the solvent accessibility in the supporting amphiphile. A notable exception is CHAPS, which yields rigid limit EPR line shapes for labels at every position along a transmembrane β-strand in OmpA. EPR line shapes from the surface of OmpA are not strongly influenced by steric interference with neighboring side chains, but are modulated by solutes that should interact with hydrophobic surfaces. These results suggest that differences in EPR spectra in different supporting environments are not the result of differences in protein dynamics but are a result of different configurations or rotameric states that are assumed by the label. This conclusion is supported by distance measurements across the OmpA β-barrel, which indicate that labels yielding more motionally restricted line shapes interact more closely with the protein surface. These results have implications for the use of spin-label-derived distance constraints in protein structure determination and demonstrate that spin labels on membrane proteins provide a highly sensitive probe for the environment surrounding a membrane protein.
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904-4319, United States.
Ricardo H Flores
RH
Jiménez
Daniel M
DM
Freed
David S
DS
Cafiso
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D002793:Cholic Acids / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D006860:Hydrogen Bonding; D008051:Lipid Bilayers / Q000737:chemistry*; D008823:Micelles; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D013113:Spin Labels
D001425:Bacterial Outer Membrane Proteins; D002793:Cholic Acids; D003902:Detergents; D008051:Lipid Bilayers; D008823:Micelles; D013113:Spin Labels; D010767:Phosphorylcholine; C078167:OMPA outer membrane proteins; C028810:dodecylphosphocholine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate