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Article

Characterization of the Hydrodynamic Properties of the Folding Transition State of an SH3 Domain by Magnetization Transfer NMR Spectroscopy^†

Martin Tollinger,^‡^§ Chris Neale,^‡^§ Lewis E. Kay,^§ and Julie D. Forman-Kay*^‡^§

Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, Department of Biochemistry and Protein Engineering Network Centres of Excellence, University of Toronto, Toronto, Ontario M5S 1A8, Canada, and Departments of Medical Genetics and Chemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Biochemistry, 2006, 45 (20), pp 6434–6445

DOI: 10.1021/bi060268o

Publication Date (Web): April 27, 2006

†

The research was supported by grants from the Canadian Institutes of Health Research (J.D.F.-K. and L.E.K.). M.T. is a recipient of an E. Schrödinger Return Fellowship of the Austrian Science Fund (R18).

‡

Hospital for Sick Children.

Department of Biochemistry and Protein Engineering Network Centres of Excellence, University of Toronto.

Current address: Department of Biomolecular Structural Chemistry, University of Vienna, Campus Vienna Biocenter 5, A-1030 Vienna, Austria.

Departments of Medical Genetics and Chemistry, University of Toronto.

To whom correspondence should be addressed. Phone: (416) 813-5358. Fax: (416) 813-5022. E-mail: forman@sickkids.ca.

Abstract

Protein folding kinetic data have been obtained for the marginally stable N-terminal Src homology 3 domain of the Drosophila protein drk (drkN SH3) in an investigation of the hydrodynamic properties of its folding transition state. Due to the presence of NMR resonances of both folded and unfolded states at equilibrium, kinetic data can be derived from NMR magnetization transfer techniques under equilibrium conditions. Kinetic analysis as a function of urea (less than 1 M) and glycerol enables determination of α values, measures of the energetic sensitivity of the transition state to the perturbation relative to the end states of the protein folding reaction (the folded and unfolded states). Both end states have previously been studied experimentally by NMR spectroscopic and other biophysical methods in great detail and under nondenaturing conditions. Combining these results with the kinetic folding data obtained here, we can characterize the folding transition state without requiring empirical models for the unfolded state structure. We are thus able to give a reliable measure of the solvent-accessible surface area of the transition state of the drkN SH3 domain (4730 ± 360 Å²) based on urea titration data. Glycerol titration data give similar results and additionally demonstrate that folding of this SH3 domain is dependent on solvent viscosity, which is indicative of at least partial hydration of the transition state. Because SH3 domains appear to fold by a common folding mechanism, the data presented here provide valuable insight into the transition states of the drkN and other SH3 domains.