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Published online before print June 2, 2006, 10.1110/ps.062136006
Protein Science (2006), 15:1769-1779. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Native state energetics of the Src SH2 domain: Evidence for a partially structured state in the denatured ensemble

David Wildes1, L. Meadow Anderson2, Alex Sabogal1 and Susan Marqusee1

1 Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA
2 Department of Chemistry, University of California, Berkeley, California 94720, USA

(RECEIVED February 6, 2006; FINAL REVISION March 27, 2006; ACCEPTED March 27, 2006)

We have defined the free-energy profile of the Src SH2 domain using a variety of biophysical techniques. Equilibrium and kinetic experiments monitored by tryptophan fluorescence show that Src SH2 is quite stable and folds rapidly by a two-state mechanism, without populating any intermediates. Native state hydrogen–deuterium exchange confirms this two-state behavior; we detect no cooperative partially unfolded forms in equilibrium with the native conformation under any conditions. Interestingly, the apparent stability of the protein from hydrogen exchange is 2 kcal/mol greater than the stability determined by both equilibrium and kinetic studies followed by fluorescence. Native-state proteolysis demonstrates that this "super protection" does not result from a deviation from the linear extrapolation model used to fit the fluorescence data. Instead, it likely arises from a notable compaction in the unfolded state under native conditions, resulting in an ensemble of conformations with substantial solvent exposure of side chains and flexible regions sensitive to proteolysis, but backbone amides that exchange with solvent ~30-fold slower than would be expected for a random coil. The apparently simple behavior of Src SH2 in traditional unfolding studies masks the significant complexity present in the denatured-state ensemble.

Keywords: hydrogen exchange; protein folding; SH2 domain; proteolysis; unfolded state; protein stability


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[Abstract] [Full Text] [PDF]


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