title: AV77 hinge mutation stabilizes the helix-turn-helix domain of trp repressor.
abstract: The structure and dynamics of the AV77 holorepressor have been studied using nuclear Overhauser enhancement spectroscopy (NOESY). By comparing NOE crosspeaks as well as proton chemical shifts, we find no evidence for any substantial difference between the wild-type and AV77 repressor structures. In addition, however, we have measured the rapid amide proton exchange rates for the DNA binding region of the apo and holo forms of the mutant and wild-type repressors using proton relaxation and saturation transfer techniques. We find that the hydrogen bonded amide protons in the DNA binding regions are stabilized for the most part by at least an order of magnitude for both forms of the mutant repressors. This is compared to a three to five fold stabilization of the holo wild-type molecule over the apo form. As the AV77 mutant is observed to be a superrepressor in vivo, we ascribe the enhanced activity of this mutant to a decrease in the instability of the DNA binding domain. We therefore suggest that the inherent instability of this domain in the wild-type molecule is needed for efficient regulation of the repressor by its corepressor, L-tryptophan, and in addition may allow for recognition of a broad range of operators.
authors.affiliation: Stanford Magnetic Resonance Laboratory, Stanford University, CA 94305-5055, USA.
authors.forename: M R
authors.initials: MR
authors.lastname: Gryk
authors.forename: O
authors.initials: O
authors.lastname: Jardetzky
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D004247:DNA; D019077:Helix-Turn-Helix Motifs; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D011522:Protons; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins
chemical_list: D001426:Bacterial Proteins; D004247:DNA; D011522:Protons; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins