Threonine 81 of the trp repressor of Escherichia coli plays an auxiliary role in the formation of the corepressor binding pocket.
A mutational study was performed on the corepressor (L-tryptophan) binding site of the trp repressor of Escherichia coli. Threonine 81, one of the residues forming the hydrophobic pocket of the binding site, was replaced with Ser, Cys and Met by cassette mutagenesis. Biochemical characterization showed that all these mutations caused a moderate decrease in tryptophan binding activity (free energy change approximately 1 kcal/mol). The results suggested that the binding pocket is rather flexible in the vicinity of Thr81. On the other hand, the mutations produced a discernible decrease in the repressor activity in vivo, apparently by weakening or eliminating the hydrogen bond between Thr81 and the operator DNA, as well as by introducing possible side-chain rearrangement.
Fujita
I
I
Department of Biological Science and Technology, Science University of Tokyo, Chiba, Japan.
Komeiji
Y
Y
Yamato
I
I
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D013912:Threonine / Q000737:chemistry* / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism*; D029968:Escherichia coli Proteins
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D012097:Repressor Proteins; D013912:Threonine; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins; C027132:1-anilino-8-naphthalenesulfonate