Reference: Haldar S, et al. (2010) Role of protein stabilizers on the conformation of the unfolded state of cytochrome c and its early folding kinetics: investigation at single molecular resolution. J Biol Chem 285(33):25314-23

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Abstract


An insight into the conformation and dynamics of unfolded and early intermediate states of a protein is essential to understand the mechanism of its aggregation and to design potent inhibitor molecules. Fluorescence correlation spectroscopy has been used to study the effects of several model protein stabilizers on the conformation of the unfolded state and early folding dynamics of tetramethyl rhodamine-labeled cytochrome c from Saccharomyces cerevisiae at single molecular resolution. Special attention has been given to arginine, which is a widely used stabilizer for improving refolding yield of different proteins. The value of the hydrodynamic radius (r(H)) obtained by analyzing the intensity fluctuations of the diffusing molecules has been found to increase in a two-state manner as the protein is unfolded by urea. The results further show that the presence of arginine and other protein stabilizers favors a relatively structured conformation of the unfolded states (r(H) of 29 A) over an extended one (r(H) of 40 A), which forms in their absence. Also, the time constant of a kinetic component (tau(R)) of about 30 micros has been observed by analyzing the correlation functions, which represents formation of a collapsed state. This time constant varies with urea concentration representing an inverted Chevron plot that shows a roll-over and behavior in the absence of arginine. To the best of our knowledge, this is one of the first applications of fluorescence correlation spectroscopy to study direct folding kinetics of a protein.

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Journal Article | Research Support, Non-U.S. Gov't
Authors
Haldar S, Mitra S, Chattopadhyay K
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