PMID:9761772
| Citation |
Frillingos, S, Sahin-Tóth, M, Wu, J and Kaback, HR (1998) Cys-scanning mutagenesis: a novel approach to structure function relationships in polytopic membrane proteins. FASEB J. 12:1281-99 |
|---|---|
| Abstract |
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. |
| Links | |
| Keywords |
Alkylation; Amino Acid Sequence; Antibodies, Monoclonal/immunology; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Binding Sites; Catalytic Domain; Cysteine; Escherichia coli/enzymology; Escherichia coli Proteins; Glutamic Acid/metabolism; Ion Transport; Membrane Proteins/chemistry; Membrane Proteins/genetics; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/genetics; Models, Molecular; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Protein Conformation; Protein Structure, Secondary; Protons; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/genetics; Structure-Activity Relationship; Symporters |
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