Native state energetics of the Src SH2 domain: Evidence for a partially structured state in the denatured ensemble

Abstract

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 Determining a protein's structure provides a wealth of information about its function, but the picture it provides is incomplete. Proteins are dynamic molecules, and under physiological conditions they can populate a range of high-energy conformations ranging from local fluctuations to completely unfolded forms. At equilibrium, the relative populations of these conformations are determined by their free energies, according to a Boltzmann distribution. These populations, and the energetic barriers to their interconversion, make up a protein's energy landscape. A complete picture of protein function demands that we understand the entire energy landscape, not just the native structure.The myriad non-native species that make up a protein's energy landscape are important for a variety of reasons. Locally distorted conformations and motions between them may play a key role in protein function, modulating enzymatic activity (Tousignant and Pelletier 2004) and allowing for allosteric regulation (Luque et al. 2002). The ability to populate an ensemble of conformations rather than a single, rigid native state also gives a single protein the flexibility needed to recognize and tightly bind a range of natural ligands (Ma et al. 2002). Such locally distorted species, however, are not the only relevant high-energy conformations in the native state ensemble of a protein; partially and globally unfolded species are also populated. While the unfolded state of a protein lacks the welldefined structure usually required to carry out specific Reprint requests to: Susan Marqusee,

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