Folding Dynamics of the src SH3 Domain ² Viara P. Grantcharova and David Baker* Department of Biochemistry, UniVersity of Washington, Seattle, Washington 98195 ReceiVed July 22, 1997; ReVised Manuscript ReceiVed September 10, 1997 X ABSTRACT: The thermodynamics and kinetics of folding of the chicken src SH3 domain were characterized using equilibrium and stopped-flow fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) hydrogen exchange experiments. As found for other SH3 domains, guanidinium chloride (GdmCl) denaturation melts followed by both fluorescence and circular dichroism were nearly superimposable, indicating the concerted formation of secondary and tertiary structure. Kinetic studies confirmed the two-state character of the folding reaction. Except for a very slow refolding phase due to proline isomerization, both folding and unfolding traces fit well to single exponentials over a wide range of GdmCl concentrations, and no burst phase in amplitude was observed during the dead time of the stopped- flow instrument. The entropy, enthalpy, and heat capacity changes upon unfolding were determined by global fitting of temperature melts at varying GdmCl concentrations (0.4-3.7 M). Estimates of the free energy of unfolding, ΔG U H 2 O , from guanidine denaturation, thermal denaturation, and kinetic experiments were in good agreement. To complement these data on the global characteristics of src SH3 folding, individual hydrogen-deuterium (HD) exchange rates were measured for approximately half of the backbone amides in 0 and 0.7 M GdmCl. The calculated free energies of the opening reaction leading to exchange (ΔG HD ) indicated that unfolding is highly cooperativesslowly exchanging protons were distributed throughout the core of the protein. The slowly exchanging protons exhibited ΔG HD values higher than the global ΔG U H 2 O by ∼1 kcal/mol, suggesting that the denatured state might be somewhat compact under native conditions. Comparison of the src SH3 with homologous SH3 domains as well as with other small well-characterized -sheet proteins provides insights into the determinants of folding kinetics and protein stability. SH3 domains have emerged in the last decade as intergal parts of many signal transduction and cytoskeletal proteins and have been shown to mediate a myriad of protein-protein interactions (1). Beside their biological importance as protein adapters, their ability to fold independently, modest size, and lack of disulfide bonds and bound cofactors make the SH3 domains an attractive model system for understanding the principles of protein folding at their simplest level. The abundance of both crystal and solution structures for many members of the SH3 family (2-10) further facilitates detailed investigations of their folding. Except for some variability in the loop regions, all structures display the distinctive SH3 fold (11): two 3-stranded -sheets packed orthogonally against each other to form a single hydrophobic core. Several SH3 domains have been the subject of detailed thermodynamic and kinetic studies to complement this structural information. Experiments on the spectrin SH3 domain (12) point to a moderately stable protein with simple two-state folding kinetics. A search for putative folding initiation sites failed to detect any peptide hairpins with nativelike structure in solution, suggesting the importance of tertiary interactions in stabilizing SH3 domains (13). Studies on the Drosophila drkN SH3 domain took advantage of the low stability of this domain to examine directly the unfolded state (14). The existence of a dynamic equilibrium between folded and unfolded protein in H 2 O enabled the application of innovative NMR 1 experiments for the detection of residual structure in the unfolded state under native conditions and in the presence of GdmCl (15, 16). Other SH3 domains studied include Sem5 SH3 (17), PI3K SH3 (J. I. Guijarro, personal communication), and human fyn SH3 (K. W. Plaxco, personal communication). The present study was motivated by two objectives. First, our laboratory has identified src SH3 mutants with highly simplified sequences that still fold properly (18). The wild- type SH3 kinetic and thermodynamic parameters reported here provide an important point of reference for the biophysical analysis of such mutants. Second, a systematic comparison between homologous SH3 domains should make apparent the spectrum of stabilities and folding rates available to this particular fold and reveal how changes in the sequence affect the parameters of folding. Such information can later be extended to comparisons with other, SH3-like proteins, which are functionally unrelated to the original members of the family: Sso7d (19), PsaE (20), and HIV-integrase DNA binding domain (21). Our results on the src SH3 domain confirm the two-state folding model for small proteins and for the first time provide residue-specific information on SH3 domain stability. Significant differences in the folding parameters of src SH3 and the other homologous proteins point to the conclusion that the determinants of the protein fold are distinct from the factors influencing stability and ² This work was supported by a grant from the Office of Naval Research (N00014-95-1-417) and Young Investigator awards to D.B. from the NSF (BIR 9512595) and the Packard Foundation (94-8453). * Corresponding author: Phone (206) 543-1295; Fax (206) 685- 1792; E-mail baker@ben.bchem.washington.edu. X Abstract published in AdVance ACS Abstracts, November 15, 1997. 1 Abbreviations: CD, circular dichroism; ESI, electrospray ionization; HD, hydrogen-deuterium; HSQC, heteronuclear single quantum coher- ence; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; GdmCl, guanidinium chloride. 15685 Biochemistry 1997, 36, 15685-15692 S0006-2960(97)01786-8 CCC: $14.00 © 1997 American Chemical Society