Spin labeling of a cysteine residue of the Escherichia coli outer membrane lipoprotein in its membrane environment.
A method was developed to attach a spin label to a specific site on the structural lipoprotein of the Escherichia coli outer membrane in situ. This method takes advantage of the fact that the outer membrane of wild-type E. coli contains few residues reactive towards sulfhydryl reagents. A mutant E. coli strain has been isolated [Suzuki, H., Nishimura, Y., Iketani, H., Campisi, J., Hirashima, A., Inouye, M. & Hirota, Y. (1976) J. Bacteriol. 127, 1494-1501] in which the second position from the carboxy terminus of the lipoprotein is changed from arginine into a cysteine residue. The membrane fraction of this mutant was treated with N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)maleimide in the presence of EDTA and 2-mercaptoethanol. Spin label was found to be preferentially incorporated into the lipoprotein. The spectrum of the spin-labeled membrane shows two components, both arising from spin label at the same site near the carboxy terminus. The strongly immobilized component has a maximum hyperfine splitting value of 53 G, and the weakly immobilized component, 37 G. A fraction of the lipoprotein is covalently bound to the peptidoglycan layer through its carboxy-terminal lysine; the spectrum of the isolated bound form of the lipoprotein was identical to that of the free form. When the matrix protein, the other major outer membrane protein, was removed by mutation, the spectrum of the lipoprotein was altered, suggesting that these two proteins are closely associated.
D001426:Bacterial Proteins; D002462:Cell Membrane / Q000648:ultrastructure; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000648:ultrastructure*; D008074:Lipoproteins; D008301:Maleimides; D008565:Membrane Proteins; D008623:Mercaptoethanol; D011485:Protein Binding; D013113:Spin Labels
D001426:Bacterial Proteins; D008074:Lipoproteins; D008301:Maleimides; D008565:Membrane Proteins; D013113:Spin Labels; D008623:Mercaptoethanol; D003545:Cysteine