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 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.
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.RonaldK@HHMI.UCLA.edu
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D000478:Alkylation; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites; D020134:Catalytic Domain; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000378:metabolism; D017136:Ion Transport; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D013329:Structure-Activity Relationship; D027981:Symporters
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011522:Protons; D011993:Recombinant Fusion Proteins; D027981:Symporters; D018698:Glutamic Acid; C021499:lactose permease; D003545:Cysteine