Nuclear magnetic resonance and fluorescence studies of substrate-induced conformational changes of histidine-binding protein J of Salmonella typhimurium.
The histidine-binding protein J of Salmonella typhimurium binds L-histidine as a first step in the high-affinity active transport of this amino acid across the cytoplasmic membrane. High-resolution nuclear magnetic resonance spectroscopy has been used to monitor the conformation of histidine-binding protein J in the presence and absence of substrate. Evidence is presented to show that this binding protein undergoes a conformational change involving a substantial number of amino-acid residues (including tryptophans) in the presence of L-histidine and that this change is specific for L-histidine. In order to monitor the involvement of tryptophan residues in the substrate-induced conformational change, 5-fluorotryptophan has been incorporated biosynthetically into the histidine-binding protein J using a tryptophan autotroph of Salmonella typhimurium. There are no significant differences in the conformation and binding activity between the 5-fluorotryptophan-labeled and the normal histidine-binding protein J. Proton and fluorine-19 nuclear magnetic resonance studies of the 5-fluorotryptophan-labeled binding protein show that at least one (and possibly two) of the tryptophan residues undergo(es) a change toward a more hydrophobic environment in the presence of L-histidine. These observations are supported by fluorescence data and by differences in the reactivity of the tryptophan residues of this protein toward N-bromosuccinimide in the presence and absence of substrate. The present results are consistent with models for the action of periplasmic-binding proteins in shock-sensitive transport systems of gram-negative bacteria which require a substrate-induced conformational change prior to the energy-dependent translocation of substrates.
D001665:Binding Sites; D001974:Bromosuccinimide; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism; D006639:Histidine / Q000378:metabolism*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis
D002352:Carrier Proteins; D008024:Ligands; D006639:Histidine; D014364:Tryptophan; D001974:Bromosuccinimide