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.
Frillingos
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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
Sahin-Tóth
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Wu
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Kaback
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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