Tollinger Martin, Neale Chris, Kay Lewis E, Forman-Kay Julie D
Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Biochemistry. 2006 May 23;45(20):6434-45. doi: 10.1021/bi060268o.
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.
在对果蝇蛋白drk的边缘稳定N端Src同源3结构域（drkN SH3）折叠过渡态的流体动力学性质进行研究时，已获得其蛋白折叠动力学数据。由于在平衡状态下同时存在折叠态和未折叠态的核磁共振共振，因此可以在平衡条件下通过核磁共振磁化转移技术获得动力学数据。作为尿素（小于约1 M）和甘油浓度函数的动力学分析能够确定α值，α值是衡量过渡态相对于蛋白质折叠反应终态（折叠态和未折叠态）对扰动的能量敏感性的指标。此前已通过核磁共振光谱法和其他生物物理方法在非变性条件下对这两种终态进行了详细的实验研究。将这些结果与此处获得的动力学折叠数据相结合，我们无需对未折叠态结构建立经验模型就能表征折叠过渡态。因此，基于尿素滴定数据，我们能够可靠地测量drkN SH3结构域过渡态的溶剂可及表面积（4730±360 Ų）。甘油滴定数据给出了类似的结果，并且进一步表明该SH3结构域的折叠依赖于溶剂粘度，这表明过渡态至少部分处于水合状态。由于SH3结构域似乎通过共同的折叠机制进行折叠，此处给出的数据为深入了解drkN和其他SH3结构域的过渡态提供了有价值的信息。
