Refolding of a small all beta-sheet protein proceeds with accumulation of kinetic intermediates.
The refolding kinetics of Cobrotoxin (CBTX), a small all beta-sheet protein is investigated using a variety of biophysical techniques including quenched-flow hydrogen-deuterium (H/D) exchange in conjunction with two-dimensional NMR spectroscopy. Urea-induced equilibrium unfolding of CBTX follows a two-state mechanism with no distinct intermediates. The protein is observed to fold very rapidly within 250 ms. Both the refolding and the unfolding limbs of the chevron plot of CBTX show a prominent curvature suggesting the accumulation of kinetic intermediates. Quenched-flow H/D exchange data suggest the presence of a broad continuum of kinetic intermediates between the unfolded and native states of the protein. Comparison of the native state hydrogen exchange data and the results of the quenched-flow H/D exchange experiments, reveals that the residues constituting the folding core of CBTX are not a subset of the slow exchange core. To our knowledge, this is the first report wherein the refolding of a small all beta-sheet protein is shown to be a multi-step process involving the accumulation of kinetic intermediates.
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.
Hui-Chu
HC
Hsieh
Thallapuranam Krishnaswamy S
TK
Kumar
Thirunavukkarasu
T
Sivaraman
Chin
C
Yu
D003039:Cobra Neurotoxin Proteins / Q000737:chemistry*; D003198:Computer Simulation; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009038:Motion; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding
D003039:Cobra Neurotoxin Proteins