Slow folding of a three-helix protein via a compact intermediate.
The KIX domain of CREB binding protein (CBP) forms a small three-helix bundle which folds autonomously. Previous equilibrium unfolding experiments led to the suggestion that folding may not be strictly two-state. To investigate the folding mechanism in more detail, the folding kinetics of KIX have been studied by urea jump fluorescence-detected stopped-flow experiments. Clear evidence for an intermediate is obtained from the plot of the natural log of the observed rate constant versus denaturant concentration, the chevron plot, and from analysis of the initial fluorescence amplitudes of the stopped-flow experiments. The chevron plot exhibits a change in shape, rollover, at low denaturant concentrations, characteristic of the formation of an intermediate. The kinetic data can be fit to a three-state model involving a compact intermediate. An on-pathway model predicts that the position of the intermediate lies close to the native state. The folding rate in the absence of denaturant is 260 s(-)(1) at pH 7.5 and 25 degrees C. This is significantly slower than the rates of other helical proteins similar in size. The slow folding may be due to the necessity of forming a buried polar interaction in the native state. The potential functional significance of the folding intermediate is discussed.
Horng
Jia-Cherng
JC
Department of Chemistry, State University of New York, Stony Brook, New York 11794-3400, USA.
Tracz
Sylvia M
SM
Lumb
Kevin J
KJ
Raleigh
Daniel P
DP
D050882:CREB-Binding Protein; D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D014508:Urea / Q000737:chemistry
D009687:Nuclear Proteins; D015534:Trans-Activators; D014508:Urea; D050882:CREB-Binding Protein; C494363:CREBBP protein, human