CYS-SCANNING MUTAGENESIS - A NOVEL-APPROACH TO STRUCTURE-FUNCTION-RELATIONSHIPS IN POLYTOPIC MEMBRANE-PROTEINS

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 res idues 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 th e 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 b y ligand binding are identified; 4) helix packing, helix tilt, and lig and-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.