Quantitative analysis of the kinetics of denaturation and renaturation of barstar in the folding transition zone.
The fluorescence-monitored kinetics of folding and unfolding of barstar by guanidine hydrochloride (GdnHCl) in the folding transition zone, at pH 7, 25 degrees C, have been quantitatively analyzed using a 3-state mechanism: U(S)<-->UF<-->N. U(S) and UF are slow-refolding and fast-refolding unfolded forms of barstar, and N is the native protein. U(S) and UF probably differ in possessing trans and cis conformations, respectively, of the Tyr 47-Pro 48 bond. The 3-state model could be used because the kinetics of folding and unfolding of barstar show 2 phases, a fast phase and a slow phase, and because the relative amplitudes of the 2 phases depend only on the final refolding conditions and not on the initial conditions. Analysis of the observed kinetics according to the 3-state model yields the values of the 4 microscopic rate constants that describe the transitions between the 3 states at different concentrations of GdnHCl. The value of the equilibrium unfolded ratio U(S):UF (K21) and the values of the rate constants of the U(S)-->UF and UF-->U(S) reactions, k12 and k21, respectively, are shown to be independent of the concentration of GdnHCl. K21 has a value of 2.1 +/- 0.1, and k12 and k21 have values of 5.3 x 10(-3) s-1 and 11.2 x 10(-3) s-1, respectively. Double-jump experiments that monitor reactions that are silent to fluorescence monitoring were used to confirm the values of K21, k12, and k21 obtained from the 3-state analysis and thereby the validity of the 3-state model.(ABSTRACT TRUNCATED AT 250 WORDS)
National Centre For Biological Sciences, TIFR Centre, Indian Institute of Science Campus, Bangalore.
M C
MC
Shastry
V R
VR
Agashe
J B
JB
Udgaonkar
D001407:Bacillus / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects; D012260:Ribonucleases / Q000037:antagonists & inhibitors*
D001426:Bacterial Proteins; D006146:Guanidines; D011994:Recombinant Proteins; C057438:barstar protein, Bacillus amyloliquefaciens; D012260:Ribonucleases; D019791:Guanidine