Three-dimensional solution NMR structure of Apo-L75F-TrpR, a temperature-sensitive mutant of the tryptophan repressor protein.
L75F-TrpR is a temperature-sensitive mutant of the tryptophan repressor protein of Escherichia coli in which surface-exposed residue leucine 75 in the DNA binding domain is replaced with phenylalanine. Biochemical and biophysical studies had suggested global alterations in dynamics for L75F-TrpR, although the structure was apparently similar to that of wild-type TrpR. Herein, we report the three-dimensional solution structure of apo-L75F-TrpR determined by multidimensional ((1)H, (15)N, and (13)C) solution NMR spectroscopy. An ensemble of structures was generated from 769 unique NOE-based distance restraints, 68 dihedral angle restraints, and 62 hydrogen bond distance restraints. Apo-L75F-TrpR exhibits a three-dimensional (3D) fold very similar to that of apo-WT-TrpR, with a dimeric core of four alpha-helices (A-C and F) from each subunit, and less well-defined D and E helical regions of the DNA binding domains. Despite their many similarities, wild-type and mutant proteins display significant chemical shift differences, one cluster of which is in the B-C turn, too distant to be ascribed solely to ring current effects from Phe75. Differences in NOE patterns and amide proton exchange rates are also observed in the B-C turn region. The data provide evidence that this point mutation exerts local effects on structure and stability in the DNA binding domain, and propagates long-range effects through the tertiary structure.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D004926:Escherichia coli; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins