Folding kinetics of a fluorescent variant of monomeric lambda repressor.
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.
Ghaemmaghami
S
S
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Word
J M
JM
Burton
R E
RE
Richardson
J S
JS
Oas
T G
TG
D019943:Amino Acid Substitution / Q000235:genetics; D010582:Bacteriophage lambda / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D004268:DNA-Binding Proteins; D019791:Guanidine; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics; D014508:Urea; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D014364:Tryptophan; D014508:Urea; D019791:Guanidine