Spin labeling of a cysteine residue of the Escherichia coli outer membrane lipoprotein in its membrane environment

N. Lee, C. Scandella, M. Inouye

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

A method was developed to attach a spin label to a specific site on the structural lipoprotein of the E. 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 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-tetramethyl-pyrrolidinyl)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.

Original languageEnglish (US)
Pages (from-to)127-130
Number of pages4
JournalProceedings of the National Academy of Sciences of the United States of America
Volume75
Issue number1
DOIs
StatePublished - 1978

All Science Journal Classification (ASJC) codes

  • General

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