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J. Biol. Chem., Vol. 282, Issue 52, 37479-37491, December 28, 2007

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Exploring the Cooperativity of the Fast Folding Reaction of a Small Protein Using Pulsed Thiol Labeling and Mass Spectrometry^*

From the National Centre for Biological Sciences, Tata Institute of Fundamental Research, Gandhi Krishi Vignana Kendra (GKVK) Campus, Bangalore 560065, India

It has been difficult to obtain directly residue-specific information on side chain packing during a fast (ms) protein folding reaction. Such information is necessary to determine the extent to which structural changes in different parts of the protein molecule are coupled together in defining the cooperativity of the overall folding transition. In this study, structural changes occurring during the major fast folding reaction of the small protein barstar have been characterized at the level of individual residue side chains. A pulsed cysteine labeling methodology has been employed in conjunction with mass spectrometry. This provides, with ms temporal resolution, direct information on structure formation at 10 different locations in barstar during its folding. Cysteine residues located on the surface of native barstar, at four different positions, remain fully solvent-accessible throughout the folding process, indicating the absence of any ephemeral nonnative structure in which these four cysteine residues get transiently buried. For buried cysteine residues, the rates of the change in cysteine-thiol accessibility to rapid chemical labeling by the thiol reagent methyl methanethiosulfonate appear to be dependent upon the location of the cysteine residue in the protein and are different from the rate measured by the change in tryptophan fluorescence. But the rates vary over only a 3-fold range. Nevertheless, a comparison of the kinetics of the change in accessibility of the cysteine 3 thiol with those of the change in the fluorescence of tryptophan 53, as well as of their denaturant dependences, indicates that the major folding reaction comprises more than one step.

Received for publication, August 13, 2007 , and in revised form, October 22, 2007.

^* This work was supported by the Tata Institute of Fundamental Research and by the Department of Biotechnology, Government of India. All mass spectra were collected at the Mass Spectrometry Facility at the National Centre for Biological Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a Shyama Prasad Mukherjee Fellowship awarded by the Council of Scientific and Industrial Research, India.

² To whom correspondence should be addressed. Fax: 91-80-23636862; E-mail: jayant{at}ncbs.res.in.

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This article has been cited by other articles:

				K. K. Sinha and J. B. Udgaonkar Barrierless evolution of structure during the submillisecond refolding reaction of a small protein PNAS, June 10, 2008; 105(23): 7998 - 8003. [Abstract] [Full Text] [PDF]