Unfolding rates of barstar determined in native and low denaturant conditions indicate the presence of intermediates.
The folding and unfolding rates of the small protein, barstar, have been monitored using stopped-flow measurements of intrinsic tryptophan fluorescence at 25 degrees C, pH 8.5, and have been compared over a wide range of urea and guanidine hydrochloride (GdnHCl) concentrations. When the logarithms of the rates of folding from urea and from GdnHCl unfolded forms are extrapolated linearly with denaturant concentration, the same rate is obtained for folding in zero denaturant. Similar linear extrapolations of rates of unfolding in urea and GdnHCl yield, however, different unfolding rates in zero denaturant, indicating that such linear extrapolations are not valid. It has been difficult, for any protein, to determine unfolding rates under nativelike conditions in direct kinetic experiments. Using a novel strategy of coupling the reactivity of a buried cysteine residue with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to the unfolding reaction of barstar, the global unfolding and refolding rates have now been determined in low denaturant concentrations. The logarithms of unfolding rates obtained at low urea and GdnHCl concentrations show a markedly nonlinear dependence on denaturant concentration and converge to the same unfolding rate in the absence of denaturant. It is shown that the native protein can sample the fully unfolded conformation even in the absence of denaturant. The observed nonlinear dependences of the logarithms of the refolding and unfolding rates observed for both denaturants are shown to be due to the presence of (un)folding intermediates and not due to movements in the position of the transition state with a change in denaturant concentration.
Sridevi
K
K
National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bangalore, India 560065.
Udgaonkar
Jayant B
JB
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry*; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods; D013439:Sulfhydryl Reagents / Q000378:metabolism; D014508:Urea / Q000737:chemistry*
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D012996:Solutions; D013439:Sulfhydryl Reagents; C057438:barstar protein, Bacillus amyloliquefaciens; D014508:Urea; D004228:Dithionitrobenzoic Acid; D019791:Guanidine; D003545:Cysteine