Ramos Carlos H I, Weisbuch Sebastien, Jamin Marc
Laboratório Nacional de Luz Síncrotron, P.O. Box 6192, Campinas SP 13084-971, Brazil.
Biochemistry. 2007 Apr 10;46(14):4379-89. doi: 10.1021/bi602574x. Epub 2007 Mar 17.
The sperm whale apomyoglobin pH 4 folding intermediate exists in two forms, Ia and Ib, that mimic transient kinetic intermediates in the folding of the native protein at pH 6. To characterize the nature of the kinetic barrier that controls the formation of the earliest intermediate Ia, we have investigated the effects of small viscogenic cosolvents on its folding and unfolding kinetics. The kinetics are measurable by stopped-flow fluorescence and follow a cooperative two-state model in the absence and presence of cosolvents. Small cosolvents stabilize Ia, but, by applying the isostability test to separate the viscogenic effect of the cosolvent from its stabilizing effect, we found that, in both folding and unfolding conditions, the apparent rate constant decreases when solvent viscosity increases. The unitary inverse dependence of the apparent rate constant on solvent viscosity indicates a diffusion-controlled reaction. This result is consistent with the hypothesis that folding of the apomyoglobin pH 4 intermediate obeys a diffusion-collision model. Additionally, the temperature dependence of the reaction rate at constant viscosity indicates that the formation of Ia is also controlled by an energy barrier. Linear free energy relationships show that the transition state of the U <==> Ia reaction is compact and buries 45% of the surface area that is buried in native apomyoglobin. We conclude that the transition state of the U <==> Ia reaction resembles that for the formation of native proteins; namely, it is dry and its compactness is closer to that of the folded (Ia) form than of the unfolded form.
抹香鲸脱辅基肌红蛋白在pH 4时的折叠中间体以两种形式存在，即Ia和Ib，它们模拟了天然蛋白在pH 6时折叠过程中的瞬态动力学中间体。为了表征控制最早中间体Ia形成的动力学屏障的性质，我们研究了小的致粘共溶剂对其折叠和去折叠动力学的影响。动力学可通过停流荧光法测量，在有无共溶剂的情况下均遵循协同二态模型。小的共溶剂使Ia稳定，但是，通过应用等稳定性测试以将共溶剂的致粘效应与其稳定效应区分开来，我们发现，在折叠和去折叠条件下，当溶剂粘度增加时，表观速率常数均降低。表观速率常数对溶剂粘度的单一反比依赖性表明这是一个扩散控制的反应。该结果与脱辅基肌红蛋白在pH 4时中间体的折叠遵循扩散碰撞模型的假设一致。此外，在恒定粘度下反应速率的温度依赖性表明Ia的形成也受能量屏障控制。线性自由能关系表明，U <==> Ia反应的过渡态是紧密的，埋藏了天然脱辅基肌红蛋白中埋藏表面积的45%。我们得出结论，U <==> Ia反应的过渡态类似于天然蛋白形成时的过渡态；也就是说，它是干燥的，其紧密程度更接近折叠（Ia）形式而非未折叠形式。
