Long-range effects on dynamics in a temperature-sensitive mutant of trp repressor

Lihua Jin, June Wong Fukayama, István Pelczer, Jannette Carey

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15 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)361-378
Number of pages18
JournalJournal of Molecular Biology
Issue number1
StatePublished - Jan 8 1999

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biophysics
  • Structural Biology


  • Adaptability
  • Ligand binding
  • Protein flexibility


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