Cys‐scanning mutagenesis: a novel approach to structure—function relationships in polytopic membrane proteins

Abstract

The entire lactose permease of Escherichia coli, a polytopic membrane transport protein that catalyzes β-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 β-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)