Long-range effects on dynamics in a temperature-sensitive mutant of trp repressor.
A mutant tryptophan repressor (TrpR) protein containing the substitution of phenylalanine for leucine 75 has been isolated following a genetic screen for temperature-sensitive mutations. Two-dimensional (2D) 1H NMR spectra indicate an overall very similar fold for the purified mutant and wild-type proteins. Circular dichroism spectropolarimetry indicates an increased helix content relative to the wild-type protein, and a slightly higher urea denaturation midpoint for the mutant protein, although there is no difference in thermal stability. Fluorescence spectra indicate a more buried environment for one or both tryptophan residues in the mutant protein. The rate of proton-deuterium exchange-out for the resolved indole ring protons of the two tryptophan residues was quantified from NMR spectra of mutant and wild-type proteins and found to be approximately 50% faster in the wild-type protein. The mutant protein binds the corepressor l-tryptophan (l-Trp) approximately ten times more weakly than does the wild-type protein, but in l-Trp excess its DNA-binding affinity is only two to fivefold weaker. Taken together the results imply that, despite its conservative chemical character and surface location at the C terminus of helix one in the helix-turn-helix DNA recognition motif, this mutational change confers long-range effects on the dynamics of the protein's secondary and tertiary structure without substantially altering its fold, and with relatively minor effects on protein function.
Jin
L
L
Chemistry Department, Princeton University, Frick Laboratory, Washington Road & William Street, Princeton, NJ, 08544-1009, USA.
Fukayama
J W
JW
Pelczer
I
I
Carey
J
J
D000577:Amides; D001426:Bacterial Proteins; D002918:Chymotrypsin; D007930:Leucine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D019906:Nuclear Magnetic Resonance, Biomolecular; D009875:Operator Regions, Genetic; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D017354:Point Mutation; D011522:Protons; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000378:metabolism; D029968:Escherichia coli Proteins
D000577:Amides; D001426:Bacterial Proteins; D002918:Chymotrypsin; D007930:Leucine; D010649:Phenylalanine; D011522:Protons; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins