Direct sugar binding to LacY measured by resonance energy transfer.
Trp151 in the lactose permease of Escherichia coli (LacY) is an important component of the sugar-binding site and the only Trp residue out of six that is in close proximity to the galactopyranoside in the structure (1PV7). The short distance between Trp151 and the sugar is favorable for Förster resonance energy transfer (FRET) to nitrophenyl or dansyl derivatives with the fluorophore at the anomeric position of galactose. Modeling of 4-nitrophenyl-alpha-d-galactopyranoside (alpha-NPG) in the binding-site of LacY places the nitrophenyl moiety about 12 A away from Trp151, a distance commensurate with the Förster distance for a Trp-nitrobenzoyl pair. We demonstrate here that alpha-NPG binding to LacY containing all six native Trp residues causes galactopyranoside-specific FRET from Trp151. Moreover, binding of alpha-NPG is sufficiently slow to resolve time-dependent fluorescence changes by stopped-flow. The rate of change in Trp --> alpha-NPG FRET is linearly dependent upon sugar concentration, which allows estimation of kinetic parameters for binding. Furthermore, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) covalently attached to the cytoplasmic end of helix X is sensitive to sugar binding, reflecting a ligand-induced conformational change. Stopped-flow kinetics of Trp --> alpha-NPG FRET and sugar-induced changes in MIANS fluorescence in the same protein reveal a two-step process: a relatively rapid binding step detected by Trp151 --> alpha-NPG FRET followed by a slower conformational change detected by a change in MIANS fluorescence.
Smirnova
Irina N
IN
Department of Physiology and Microbiology, Immunology & Molecular Genetics, Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 90095-7327, USA.
Kasho
Vladimir N
VN
Kaback
H Ronald
HR
D019943:Amino Acid Substitution / Q000235:genetics; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D008024:Ligands; D008956:Models, Chemical; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009578:Nitrobenzenes / Q000378:metabolism; D011487:Protein Conformation; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D013862:Thiogalactosides / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
D000817:Anilino Naphthalenesulfonates; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D009004:Monosaccharide Transport Proteins; D009578:Nitrobenzenes; D027981:Symporters; D013862:Thiogalactosides; C006173:dansylgalactoside; C016014:4-nitrophenylglycerol; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D014364:Tryptophan