Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines.
Site-directed mutagenesis was used to produce mutants of bacteriorhodopsin where either glycine-72, threonine-90, leucine-92, or serine-169 was replaced by a cysteine. Two different spin labels were then covalently attached to these sites. The selection of attachment sites covered two postulated loops (72,169) and a membrane-spanning segment (90,92). It was not possible to properly refold the protein labeled at position 90, presumably due to steric problems, but the EPR spectra of the other mutants that were successfully reconstituted in phospholipid vesicles provided information on the dynamics of protein side chains in the vicinity of the label site. A power saturation approach was used to investigate the spin relaxation times, which in turn can be influenced by collisions with paramagnetic species. The differential effect of oxygen and a water-soluble chromium complex on the power-saturation behavior of the spin-labeled mutants was used to obtain topographical information on the sites in the membrane-bound protein. The results are consistent with residues 72 and 169 being located in structured loops exposed to the aqueous phase and residue 92 being localized in the membrane interior, possibly near a helix-helix contact region.
Altenbach
C
C
Jules Stein Eye Institute, University of California, Los Angeles 90024-1771.
Flitsch
S L
SL
Khorana
H G
HG
Hubbell
W L
WL
D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D002462:Cell Membrane / Q000032:analysis; D003545:Cysteine / Q000032:analysis*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004926:Escherichia coli / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics; D009154:Mutation; D009589:Nitrogen Oxides; D011487:Protein Conformation; D013113:Spin Labels
D001426:Bacterial Proteins; D008565:Membrane Proteins; D009589:Nitrogen Oxides; D013113:Spin Labels; C039900:nitroxyl; D003545:Cysteine