Cysteine 148 in the lactose permease of Escherichia coli is a component of a substrate binding site. 2. Site-directed fluorescence studies.
By using site-directed fluorescence spectroscopy, we have carried out structure/function studies on lactose permease purified from Escherichia coli in dodecyl beta, D-maltoside. Initially, permease containing a single native Cys at position 148 (helix V) was studied, since this residue is protected against alkylation by substrates of the permease. In the absence of ligand, Cys 148 permease reacts rapidly with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS), a fluorophore whose quantum yield increases dramatically upon reaction with a thiol, indicating that this residue is readily accessible to the probe. Various ligands of the permease block the reaction, and the concentration dependence is commensurate with the affinity of each ligand for the permease (i.e., beta, D-galactopyranosyl 1-thio-beta, D-galactopyranoside << lactose < galactose), but neither sucrose nor glucose has any effect whatsoever. Thus, the permease retains the ability to bind ligand specifically when the molecule is in dodecyl beta, D-maltoside. Permease containing single Cys substitutions in the vicinity of Cys 148 was also studied. Interestingly, labeling of Cys 145 which is presumed to be one helical turn removed from Cys 148 exhibits properties similar to those observed with Cys 148 permease, but the effects of ligand are far less dramatic. On the other hand, permease with a single Cys residue at position 146 or 147 behaves in a completely different manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Wu
J
J
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1662.
Kaback
H R
HR
D000817:Anilino Naphthalenesulfonates; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005690:Galactose / Q000378:metabolism; D007785:Lactose / Q000378:metabolism; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents; D027981:Symporters; D014312:Trisaccharides / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013439:Sulfhydryl Reagents; D027981:Symporters; D014312:Trisaccharides; C078115:glucosyl-O-galactosyl-(1-4)glucoside; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; C021499:lactose permease; D007785:Lactose; D003545:Cysteine; D005690:Galactose