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Intermolecular thiol cross-linking via loops in the lactose permease of Escherichia coli.

ABSTRACT: Previous experiments using intermolecular thiol cross-linking to determine surface-exposed positions in the transmembrane helices of the lactose permease suggest that only positions accessible from the aqueous phase are susceptible to cross-linking. This approach is now extended to most of the remaining positions in the molecule. Of an additional 143 single-Cys mutants studied, homodimer formation is observed with both a 5-A- and a 21-A-long crosslinking agent containing bis-methane thiosulfonate reactive groups in 33 mutants and exclusively with the 21-A-long reagent in 43 mutants. Furthermore, intermolecular cross-linking has little or no effect on transport activity, thereby providing further support for the argument that lactose permease is functionally, as well as structurally, a monomer in the membrane. In addition, evidence is presented indicating that reentrance loops are unlikely in this polytopic membrane transport protein.

SUBMITTER: Ermolova N

PROVIDER: S-EPMC193537 | biostudies-literature | 2003 Sep

REPOSITORIES: biostudies-literature

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Intermolecular thiol cross-linking via loops in the lactose permease of Escherichia coli.

Ermolova Natalia N Guan Lan L Kaback H Ronald HR

Proceedings of the National Academy of Sciences of the United States of America 20030821 18

Previous experiments using intermolecular thiol cross-linking to determine surface-exposed positions in the transmembrane helices of the lactose permease suggest that only positions accessible from the aqueous phase are susceptible to cross-linking. This approach is now extended to most of the remaining positions in the molecule. Of an additional 143 single-Cys mutants studied, homodimer formation is observed with both a 5-A- and a 21-A-long crosslinking agent containing bis-methane thiosulfonat ...[more]

PMID: 12934015

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