Barrierless evolution of structure during the submillisecond refolding reaction of a small protein.
To determine whether a protein folding reaction can occur in the absence of a dominant barrier is crucial for understanding its complexity. Here direct ultrafast kinetic measurements have been used to study the initial submillisecond (sub-ms) folding reaction of the small protein barstar. The cooperativity of the initial folding reaction has been explored by using two probes: fluorescence resonance energy transfer, through which the contraction of two intramolecular distances is measured, and the binding of 8-anilino-1-naphthalene sulfonic acid, through which the formation of hydrophobic clusters is monitored. A fast chain contraction is shown to precede the formation of hydrophobic clusters, indicating that the sub-ms folding reaction is not cooperative. The observed rate constant of the sub-ms folding reaction monitored by 8-anilino-1-naphthalene sulfonic acid fluorescence has been found to be the same in stabilizing conditions (low urea concentrations), in which specific structure is formed, and in marginally stabilizing conditions (higher urea concentrations), where virtually no structure is formed in the product of the sub-ms folding reaction. The observation that the folding rate is independent of the folding conditions suggests that the initial folding reaction occurs in the absence of a dominant free energy barrier. These results provide kinetic evidence that the formation of specific structure need not be slowed down by any significant free energy barrier during the course of a very fast protein folding reaction.
Sinha
Kalyan K
KK
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India.
Udgaonkar
Jayant B
JB
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D019143:Evolution, Molecular; D007700:Kinetics; D017510:Protein Folding; D013816:Thermodynamics; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; C057438:barstar protein, Bacillus amyloliquefaciens; D014508:Urea