Khan Faaizah, Chuang Jessica I, Gianni Stefano, Fersht Alan R
MRC Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge CB2 2QH, UK.
J Mol Biol. 2003 Oct 10;333(1):169-86. doi: 10.1016/j.jmb.2003.08.024.
To search for folding intermediates, we have examined the folding and unfolding kinetics of wild-type barnase and four representative mutants under a wide range of conditions that span two-state and multi-state kinetics. The choice of mutants and conditions provided in-built controls for artifacts that might distort the interpretation of kinetics, such as the non-linearity of kinetic and equilibrium data with concentration of denaturant. We measured unfolding rate constants over a complete range of denaturant concentration by using by 1H/2H-exchange kinetics under conditions that favour folding, conventional stopped-flow methods at higher denaturant concentrations and continuous flow. Under conditions that favour multi-state kinetics, plots of the rate constants for unfolding against denaturant concentration fitted quantitatively to the equation for three-state kinetics, with a sigmoid component for a change of rate determining step, as did the refolding kinetics. The position of the transition state on the reaction pathway, as measured by solvent exposure (the Tanford beta value) also moved with denaturant concentration, fitting quantitatively to the same equations with a change of rate determining step. The sigmoid behaviour disappeared under conditions that favoured two-state kinetics. Those data combined with direct structural observations and simulation support a minimal reaction pathway for the folding of barnase that involves two detectable folding intermediates. The first intermediate, I(1), is the denatured state under physiological conditions, D(Phys), which has native-like topology, is lower in energy than the random-flight denatured state U and is suggested by molecular dynamics simulation of unfolding to be on-pathway. The second intermediate, I(2), is high energy, and is proven by the change in rate determining step in the unfolding kinetics to be on-pathway. The change in rate determining step in unfolding with structure or environment reflects the change in partitioning of this intermediate to products or starting materials.
为了寻找折叠中间体，我们研究了野生型核糖核酸酶 barnase 和四个代表性突变体在跨越两态和多态动力学的广泛条件下的折叠和去折叠动力学。突变体和条件的选择为可能扭曲动力学解释的假象提供了内在对照，例如动力学和平衡数据随变性剂浓度的非线性。我们通过在有利于折叠的条件下使用 1H/2H 交换动力学、在较高变性剂浓度下使用传统的停流方法以及连续流动，测量了整个变性剂浓度范围内的去折叠速率常数。在有利于多态动力学的条件下，去折叠速率常数对变性剂浓度的图定量拟合三态动力学方程，具有速率决定步骤变化的 S 形成分，重折叠动力学也是如此。通过溶剂暴露测量的反应途径上过渡态的位置（Tanford β 值）也随变性剂浓度移动，定量拟合相同方程且速率决定步骤发生变化。在有利于两态动力学的条件下，S 形行为消失。这些数据与直接的结构观察和模拟相结合，支持了 barnase 折叠的最小反应途径，该途径涉及两个可检测的折叠中间体。第一个中间体 I(1) 是生理条件下的变性态 D(Phys)，具有类似天然的拓扑结构，能量低于随机飞行变性态 U，并且通过去折叠的分子动力学模拟表明其处于反应途径上。第二个中间体 I(2) 能量较高，并且通过去折叠动力学中速率决定步骤的变化证明其处于反应途径上。去折叠过程中速率决定步骤随结构或环境的变化反映了该中间体分配到产物或起始材料的变化。
