19F NMR study of the leucine-specific binding protein of Escherichia coli: mutagenesis and assignment of the 5-fluorotryptophan-labeled residues.
The Escherichia coli L-leucine receptor is an aqueous protein and the first component in the distinct transport pathway for hydrophobic amino acids. L-leucine binding induces a conformational change, which enables the receptor to dock to the membrane components. To investigate the ligand-induced conformational change and binding properties of this protein, we used (19)F NMR to probe the four tryptophan residues located in the two lobes of the protein. The four tryptophan residues were labeled with 5-fluorotryptophan and assigned by site-directed mutagenesis. The (19)F NMR spectra of the partially ligand free proteins show broadened peaks which sharpen when L-leucine is bound, showing that the labeled wild-type protein and mutants are functional. The titration of L-phenylalanine into the 5-fluorotryptophan labeled wild-type protein shows the presence of closed and open conformers. Urea-induced denaturation studies support the NMR results that the wild-type protein binds L-phenylalanine in a different manner to L-leucine. Our studies showed that the tryptophan to phenylalanine mutations on structural units linked to the binding pocket produce subtle changes in the environment of Trp18 located directly in the binding cleft.
Salopek-Sondi
Branka
B
Department of Chemistry, Clarkson University, Potsdam, NY 13699, USA.
Luck
Linda A
LA
D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins; D007930:Leucine / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D016296:Mutagenesis; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000235:genetics; D010649:Phenylalanine / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D013194:Staining and Labeling; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000378:metabolism*
D029968:Escherichia coli Proteins; C024913:LivK protein, E coli; D033902:Periplasmic Binding Proteins; C026927:5-fluorotryptophan; D010649:Phenylalanine; D014364:Tryptophan; D007930:Leucine