Wani Ajazul Hamid, Udgaonkar Jayant B
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India.
J Mol Biol. 2009 Mar 27;387(2):348-62. doi: 10.1016/j.jmb.2009.01.060. Epub 2009 Feb 4.
Kinetic and equilibrium studies of the folding and unfolding of the SH3 domain of the PI3 kinase, have been used to identify a folding intermediate that forms after the rate-limiting step on the folding pathway. Folding and unfolding, in urea as well as in guanidine hydrochloride (GdnHCl), were studied by monitoring changes in the intrinsic fluorescence or in the far-UV circular dichroism (CD) of the protein. The two probes yield non-coincident equilibrium transitions for unfolding in urea, indicating that an intermediate, I, exists in equilibrium with native (N) and unfolded (U) protein, during unfolding. Hence, the equilibrium unfolding data were analyzed according to a three-state N <--> I <--> U mechanism. An intermediate is observed also in kinetic unfolding studies, and its presence leads to the unfolding reaction in urea as well as in GdnHCl, occurring in two steps. The fast step is complete within the initial 11 ms of unfolding and manifests itself in a burst phase change in fluorescence. At high concentrations of GdnHCl, the entire change in fluorescence during unfolding occurs during the 11 ms burst phase. CD measurements indicate, however, that I retains N-like secondary structure. An analysis of the kinetic and thermodynamic data, according to a minimal three-state N <--> I <--> U mechanism, positions I after the rate-limiting transition state, TS1, of folding, on the reaction coordinate of folding in GdnHCl. Hence, I is not revealed when folding is commenced from U, regardless of the nature of the probe used to follow the folding reaction. Interrupted unfolding experiments, in which the protein is unfolded transiently in GdnHCl for various lengths of time before being refolded, showed that I refolds to N much faster than does U, confirms the analysis of the direct folding and unfolding experiments, that I is formed after the rate-limiting step of refolding in GdnHCl.
对PI3激酶SH3结构域折叠与去折叠的动力学及平衡研究，已用于识别在折叠途径中限速步骤之后形成的折叠中间体。通过监测蛋白质固有荧光或远紫外圆二色性（CD）的变化，研究了在尿素以及盐酸胍（GdnHCl）中的折叠与去折叠过程。这两种探针在尿素中去折叠时产生不重合的平衡转变，表明在去折叠过程中，中间体I与天然态（N）和去折叠态（U）蛋白质处于平衡状态。因此，根据三态N <--> I <--> U机制对平衡去折叠数据进行了分析。在动力学去折叠研究中也观察到了中间体，其存在导致在尿素以及GdnHCl中的去折叠反应分两步进行。快速步骤在去折叠的最初11毫秒内完成，并表现为荧光的突发相变化。在高浓度GdnHCl下，去折叠过程中荧光的整个变化都发生在11毫秒的突发相中。然而，CD测量表明I保留了类似N的二级结构。根据最小三态N <--> I <--> U机制对动力学和热力学数据进行分析，在GdnHCl中折叠的反应坐标上，I位于折叠的限速过渡态TS1之后。因此，当从U开始折叠时，无论用于跟踪折叠反应的探针性质如何，都不会揭示I。中断去折叠实验中，蛋白质先在GdnHCl中短暂去折叠不同时长，然后再重新折叠，结果表明I重新折叠为N的速度比U快得多，这证实了对直接折叠和去折叠实验的分析，即I是在GdnHCl中重新折叠的限速步骤之后形成的。
