Bezsonova Irina, Korzhnev Dmitry M, Prosser R Scott, Forman-Kay Julie D, Kay Lewis E
Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6.
Biochemistry. 2006 Apr 18;45(15):4711-9. doi: 10.1021/bi060177r.
The volumetric properties associated with protein folding transitions reflect changes in protein packing and hydration of the states that participate in the folding reaction. Here, NMR spin relaxation techniques are employed to probe the folding-unfolding kinetics of two SH3 domains as a function of pressure so that the changes in partial molar volumes along the folding pathway can be measured. The two domains fold with rates that differ by approximately 3 orders of magnitude, so their folding dynamics must be probed using different NMR relaxation experiments. In the case of the drkN SH3 domain that folds via a two-state mechanism on a time scale of seconds, nitrogen magnetization exchange spectroscopy is employed, while for the G48M mutant of the Fyn SH3 domain where the folding occurs on the millisecond time scale (three-step reaction), relaxation dispersion experiments are utilized. The NMR methodology is extremely sensitive to even small changes in equilibrium and rate constants, so reliable estimates of partial molar volumes can be obtained using low pressures (1-120 bar), thus minimizing perturbations to any of the states along the folding reaction coordinate. The volumetric data that were obtained are consistent with a similar folding mechanism for both SH3 domains, involving early chain compaction to states that are at least partially hydrated. This work emphasizes the role of NMR spin relaxation in studying dynamic processes over a wide range of time scales.
与蛋白质折叠转变相关的体积性质反映了参与折叠反应的各个状态下蛋白质堆积和水合作用的变化。在此，我们运用核磁共振自旋弛豫技术来探究两个SH3结构域的折叠-去折叠动力学随压力的变化情况，以便能够测量沿折叠途径的偏摩尔体积变化。这两个结构域的折叠速率相差约3个数量级，因此必须使用不同的核磁共振弛豫实验来探测它们的折叠动力学。对于通过双态机制在秒级时间尺度上折叠的drkN SH3结构域，采用氮磁化交换光谱法；而对于Fyn SH3结构域的G48M突变体，其折叠发生在毫秒时间尺度上（三步反应），则利用弛豫色散实验。核磁共振方法对平衡常数和速率常数的微小变化极为敏感，因此可以在低压（1 - 120巴）下获得可靠的偏摩尔体积估计值，从而将对折叠反应坐标上任何状态的扰动降至最低。所获得的体积数据与两个SH3结构域类似的折叠机制一致，即早期链压缩形成至少部分水合的状态。这项工作强调了核磁共振自旋弛豫在研究广泛时间尺度上的动态过程中的作用。
