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Structure is lost incrementally during the unfolding of barstar

Nature Structural Biology volume 8, pages 799804 (2001) | Download Citation

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Abstract

Coincidental equilibrium unfolding transitions observed by multiple structural probes are taken to justify the modeling of protein unfolding as a two-state, N U, cooperative process. However, for many of the large number of proteins that undergo apparently two-state equilibrium unfolding reactions, folding intermediates are detected in kinetic experiments. The small protein barstar is one such protein. Here the two-state model for equilibrium unfolding has been critically evaluated in barstar by estimating the intramolecular distance distribution by time-resolved fluorescence resonance energy transfer (TR-FRET) methods, in which fluorescence decay kinetics are analyzed by the maximum entropy method (MEM). Using a mutant form of barstar containing only Trp 53 as the fluorescence donor and a thionitrobenzoic acid moiety attached to Cys 82 as the fluorescence acceptor, the distance between the donor and acceptor has been shown to increase incrementally with increasing denaturant concentration. Although other probes, such as circular dichroism and fluorescence intensity, suggest that the labeled protein undergoes two-state equilibrium unfolding, the TR-FRET probe clearly indicates multistate equilibrium unfolding. Native protein expands progressively through a continuum of native-like forms that achieve the dimensions of a molten globule, whose heterogeneity increases with increasing denaturant concentration and which appears to be separated from the unfolded ensemble by a free energy barrier.

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Acknowledgements

We thank N. Periasamy for the MEM software and advice on its use; A.S.R. Koti for discussion and help regarding MEM analysis; and M.K. Mathew, S. Mayor and B. Rami for critical reading of the manuscript. This work was funded by the Tata Institute of Fundamental Research and by the Wellcome Trust. J.B.U. is the recipient of a Swarnajayanti Fellowship from the Government of India.

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  1. Department of Chemical Sciences, Tata Institute of Fundamental Research Mumbai 400005, India.

    • G.S. Lakshmikanth
    •  & G. Krishnamoorthy
  2. National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India.

    • K. Sridevi
    •  & Jayant B. Udgaonkar

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Correspondence to G. Krishnamoorthy or Jayant B. Udgaonkar.

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DOI

https://doi.org/10.1038/nsb0901-799

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