Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a Reprint requests to: Kevin W. Plaxco, Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA; e-mail: kwp@chem.ucsb.edu; fax: (805) 893-4120.Abbreviations: GuHCl, guanidine hydrochloride; tris, tris hydroxymethylaminoethane; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TCEP, tris(2-carboxyethyl)phosphine; CD, circular dichroism. Article published online ahead of print. Article and publication date are at

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,

Amide hydrogen-deuterium exchange has proven to be a powerful tool for detecting and characterizing high-energy conformations in protein ensembles. Since interactions with ligands can modulate these highenergy conformations, hydrogen exchange appears to be an ideal experimental probe of the physical mechanisms underlying processes like allosteric regulation. The chemical mechanism of hydrogen exchange, however, can complicate such studies. Here, we examine hydrogen exchange rates in a simple model system, the c-Src SH3 domain interacting with a short peptide ligand. Addition of ligand slows the rates of hydrogen exchange at nearly every amide for which we can obtain data. Careful analysis, however, reveals that this slowing is due primarily to a reduction in the population of free protein in the system, and not to any specific property of the complex. We present a method to separate the contributions of free and bound protein to the exchange kinetics that has allowed us to identify the subset of amides where exchange arises directly from the complex. These results demonstrate that the slowing of hydrogen exchange induced by ligand interactions should be interpreted with caution, and more extensive experiments are required to correlate changes in hydrogen exchange with changes in structure or internal dynamics.Keywords: SH3; hydrogen exchange; ligand binding; protein dynamics Proteins are highly dynamic molecules. A variety of experimental techniques have revealed protein motions ranging from sidechain rotations to large domain rearrangements, on timescales from nanoseconds to years. Some of these motions can be thought of as transitions between the most populated, native conformation and less populated highenergy states. Under conditions that favor the native conformations, myriad high-energy forms are also sampled, ranging from small local fluctuations to complete unfolding. At equilibrium, the relative population of each conformation is determined by its energy according to a Boltzmann distribution.Although the existence of high-energy forms in the native state ensemble is well established, the functional significance of these conformations in many proteins remains unclear. In allosteric regulation, they may be extremely important. Models of allostery postulate an equilibrium between inactive, tense states and high-energy, active, relaxed states (Monod et al. 1965;Koshland et al. 1966). Positive effectors preferentially bind the active ensemble, shifting the equilibrium and activating the enzyme. This model is well recognized for large, multimeric proteins like hemoglobin and allosteric enzymes. In addition, phosphorylation of the bacterial signaling molecule NtrC has been shown to alter a preexisting equilibrium between low-and high-energy states, favoring active conformations (Volkman et al. 2001). Computer modeling has implicated a similar mechanism for allosteric control in the monomeric enzyme dihydrofolate reductase (Pan et al. 2000), and has suggested that this mechanism may be broadly applicabl...

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