Solution Structure, Dynamics, and Thermodynamics of the Native State Ensemble of the Sem-5 C-Terminal SH3 Domain ² Josephine C. Ferreon, David E. Volk, Bruce A. Luxon, David G. Gorenstein, and Vincent J. Hilser* Department of Human Biological Chemistry and Genetics and Sealy Center for Structural Biology, UniVersity of Texas Medical Branch at GalVeston, GalVeston, Texas 77555-1055 ReceiVed January 3, 2003; ReVised Manuscript ReceiVed March 18, 2003 ABSTRACT: Although the high-resolution structure of a protein may provide significant insight into which regions are important for function, it is well-known that proteins undergo significant conformational fluctuations, even under native conditions. This suggests that the static structure alone may not provide sufficient information for elucidation of the thermodynamic determinants of biological function and that an accurate molecular-level description of function requires knowledge of the nature and energetics of the conformational states that constitute the native state ensemble. Here the native state ensemble of the C-terminal src homology domain-3 (C-SH3) from Caenorhabditis elegans Sem-5 has been studied using a variety of high-resolution biophysical techniques. In addition to determining the first solution structure of the unliganded protein, we have performed 15 N relaxation and native state hydrogen-deuterium exchange. It is observed that the regions of greatest structural variabilility also show low protection and order parameters, suggesting a higher degree of conformational diversity. These flexible regions also coincide with those regions of Sem-5 that have been predicted by the COREX algorithm to be unfolded in many of the most probable conformational states within the native state ensemble. The implications of this agreement and the potential role of conformational heterogeneity of the observed biophysical properties are discussed. The existence of conformational excursions in proteins is well-established from hydrogen-deuterium (H/D) 1 exchange and NMR relaxation experiments (1-5). Despite knowledge of their presence, the extent of motion and the motional pathways have only been marginally characterized. In ensemble terms, little is known of the detailed structural and thermodynamic attributes of the relevant microscopic states comprising the native state ensemble. The importance of understanding the structural and thermodynamic details of the conformational states that exist under native conditions cannot be overstated. Because the observed biological activity of a particular protein results from the energy (or Boltz- mann)-weighted contributions of the component microstates in the ensemble, knowledge of the structural and thermo- dynamic characteristics of these states is a prerequisite for a molecular-level understanding of protein function. Most of what is known to date about the energetics of the native state conformational ensemble comes from native state amide H/D exchange experiments. According to these analyses, which have been performed on numerous proteins, most backbone amides within a particular protein become exposed to solvent, and therefore exchange competent, as a result of local unfolding reactions that are many times more probable than global unfolding under native conditions (6). While these experiments clearly demonstrate that the equi- librium under such conditions is more heavily influenced by local unfolding fluctuations than by global unfolding, a structural and thermodynamic characterization of the states comprising these fluctuations has proven to be problematic for several reasons. First, because hydrogen exchange does not provide the simultaneity of the exchange reactions, the cooperativity cannot be unambiguously assigned. Second, and equally important, the exchange rates of many amides are extremely high in typical H/D exchange experiments such that exchange is completed during the “dead time” of the experiment. As a consequence of this limitation, states with free energies of less than ∼3.0 kcal/mol (i.e., states with the highest probability) cannot be detected by standard H/D exchange experiments. Here we utilize a wide range of high-resolution biophysical techniques to address the structure and energetics of the native state conformational ensemble of the C-terminal SH3 (C-SH3) domain of the Caenorhabditis elegans protein, Sem- 5. In particular, we focus on the extent to which the component microstates in the macroscopic native state deviate both structurally and thermodynamically from the average structure. In addition to determining the 15 N relax- ation rates, and H/D exchange rates, we have determined the first high-resolution structure of the unligated protein using multidimensional NMR. The experimental results ² Supported by National Science Foundation Grant MCB-9875689, National Institutes of Health Grant R01-GM13747, and Welch Award H-1461. J.C.F. is the recipient of a Sealy Center for Structural Biology Pre-Doctoral Fellowship. * To whom correspondence should be addressed. Fax: (409) 747- 6816. Telephone: (409) 747-6812. E-mail: vince@hbcg.utmb.edu. 1 Abbreviations: SH3, src-homology domain 3; C-SH3, C-terminal SH3 domain; Sos, Son of Sevenless; mSos, murine Sos; 2D, two- dimensional; 3D, three-dimensional; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser enhancement; HSQC, heteronuclear single- quantum coherence; NOESY, NOE spectroscopy; rmsd, root-mean- square deviation; H-bond, hydrogen bond; H/D, hydrogen-deuterium. 5582 Biochemistry 2003, 42, 5582-5591 10.1021/bi030005j CCC: $25.00 © 2003 American Chemical Society Published on Web 04/25/2003