title: Characterization of the hydrodynamic properties of the folding transition state of an SH3 domain by magnetization transfer NMR spectroscopy.
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 approximately 1 M) and glycerol enables determination of alpha 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 A(2)) 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.
authors.affiliation: Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
authors.forename: Martin
authors.initials: M
authors.lastname: Tollinger
authors.forename: Chris
authors.initials: C
authors.lastname: Neale
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
authors.forename: Julie D
authors.initials: JD
authors.lastname: Forman-Kay
mesh_terms: D000818:Animals; D004330:Drosophila / Q000737:chemistry / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D011489:Protein Denaturation; D017510:Protein Folding; D012997:Solvents / Q000378:metabolism; D013816:Thermodynamics; D013997:Time Factors; D017186:Titrimetry; D014508:Urea / Q000378:metabolism; D014783:Viscosity; D018909:src Homology Domains
chemical_list: D029721:Drosophila Proteins; D012997:Solvents; C080530:drk protein, Drosophila; D014508:Urea; D005990:Glycerol