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

^a Howard Hughes Medical Institute, Departments of Physiology and Microbiology and Molecular Genetics, Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 90024–1570

The entire lactose permease of Escherichia coli, a polytopic membrane transport protein that catalyzes ß-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 ß-galactoside and H⁺ translocation.—Frillingos, S., Sahin-Tóth, M., Wu, J., Kabac, H. R. Cys-scanning mutagenesis: a novel approach to structure-function relationships in polytopic membrane proteins. FASEB J. 12, 1281–1299 (1998)

Key Words: active transport • bioenergetics • membrane protein structure • site-directed mutagenesis • ligand binding • conformational change

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