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J. Biol. Chem., Vol. 279, Issue 39, 40303-40313, September 24, 2004

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Osmolytes Induce Structure in an Early Intermediate on the Folding Pathway of Barstar^*

Lovy Pradeep and Jayant B. Udgaonkar

From the National Centre for Biological Sciences, Tata Institute of Fundamental Research, Gandhi Krishi Vigyan Kendra Campus, Bangalore 560 065, India

Osmolytes stabilize proteins against denaturation, but little is known about how their stabilizing effect might affect a protein folding pathway. Here, we report the effects of the osmolytes, trimethylamine-N-oxide, and sarcosine on the stability of the native state of barstar as well as on the structural heterogeneity of an early intermediate ensemble, I_E, on its folding pathway. Both osmolytes increase the stability of the native protein to a similar extent, with stability increasing linearly with osmolyte concentration. Both osmolytes also increase the stability of I_E but to different extents. Such stabilization leads to an acceleration in the folding rate. Both osmolytes also alter the structure of I_E but do so differentially; the fluorescence and circular dichroism properties of I_E differ in the presence of the different osmolytes. Because these properties also differ from those of the unfolded form in refolding conditions, different burst phase changes in the optical signals are seen for folding in the presence of the different osmolytes. An analysis of the urea dependence of the burst phase changes in fluorescence and circular dichroism demonstrates that the formation of I_E is itself a multistep process during folding and that the two osmolytes act by stabilizing, differentially, different structural components present in the I_E ensemble. Thus, osmolytes can alter the basic nature of a protein folding pathway by discriminating, through differential stabilization, between different members of an early intermediate ensemble, and in doing so, they thereby appear to channel folding along one route when many routes are available.

Received for publication, June 7, 2004 , and in revised form, July 16, 2004.

^* This work was funded by the Tata Institute of Fundamental Research and a grant from the Department of Science and Technology of the Government of India. 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.

Recipient of a Swarnajayanti Fellowship from the Government of India. To whom correspondence should be addressed. Tel.: 91-80-23636420; Fax: 91-80-23636662; E-mail: jayant{at}ncbs.res.in.

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