Folding kinetics of a fluorescent variant of monomeric lambda repressor

Biochemistry. 1998 Jun 23;37(25):9179-85. doi: 10.1021/bi980356b.


A tryptophan-containing variant of monomeric lambda repressor has been made, and its folding kinetics were analyzed at 20 degreesC using fluorescence stopped-flow and dynamic NMR. Equilibrium denaturation curves obtained by circular dichroism, fluorescence, and NMR are superimposable. Stopped-flow analysis indicates that in the absence of denaturants the folding reaction is complete within the dead-time of the experiment. Within higher denaturant conditions, where the folding rate is slower, NMR and stopped-flow agree on the folding and unfolding rates of the protein. In 3.4 M urea and 1.8 M GdmCl, we show that the variant folds within 2 ms. Extrapolation indicates that the folding time is 20 micro(s) in the absence of denaturants. All folding and unfolding reactions displayed monoexponential kinetics, and no burst-phases were observed. In addition, the thermodynamic parameters Delta G and meq obtained from the kinetic analysis are consistent with the equilibrium experiments. The results support a two-state Dleft and right arrow N folding model.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Substitution / genetics
  • Bacteriophage lambda / chemistry*
  • Bacteriophage lambda / genetics
  • Circular Dichroism
  • DNA-Binding Proteins*
  • Guanidine
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Protein Denaturation
  • Protein Folding*
  • Repressor Proteins / chemistry*
  • Repressor Proteins / genetics*
  • Repressor Proteins / metabolism
  • Spectrometry, Fluorescence
  • Tryptophan / genetics
  • Urea
  • Viral Proteins
  • Viral Regulatory and Accessory Proteins


  • DNA-Binding Proteins
  • Repressor Proteins
  • Viral Proteins
  • Viral Regulatory and Accessory Proteins
  • phage repressor proteins
  • Tryptophan
  • Urea
  • Guanidine