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FASEB J. 1998 Oct;12(13):1281-99.

Cys-scanning mutagenesis: a novel approach to structure function relationships in polytopic membrane proteins.

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  • 1Howard Hughes Medical Institute, Departments of Physiology and Microbiology and Molecular Genetics, Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 90024 1570.RonaldK@HHMI.UCLA.edu

Abstract

The entire lactose permease of Escherichia coli, a polytopic membrane transport protein that catalyzes beta-galactoside/H+ symport, has been subjected to Cys-scanning mutagenesis in order to determine which residues play an obligatory role in the mechanism and to create a library of mutants with a single-Cys residue at each position of the molecule for structure/function studies. Analysis of the mutants has led to the following: 1) only six amino acid side chains play an irreplaceable role in the transport mechanism; 2) positions where the reactivity of the Cys replacement is increased upon ligand binding are identified; 3) positions where the reactivity of the Cys replacement is decreased by ligand binding are identified; 4) helix packing, helix tilt, and ligand-induced conformational changes are determined by using the library of mutants in conjunction with a battery of site-directed techniques; 5) the permease is a highly flexible molecule; and 6) a working model that explains coupling between beta-galactoside and H+ translocation. structure-function relationships in polytopic membrane proteins.

PMID:
9761772
[PubMed - indexed for MEDLINE]
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