Tan Y J, Oliveberg M, Fersht A R
Cambridge Centre for Protein Engineering, UK.
J Mol Biol. 1996 Nov 29;264(2):377-89. doi: 10.1006/jmbi.1996.0647.
CI2 folds and unfolds as a single cooperative unit by simple two-state kinetics, which enables the properties of the transition state to be measured from both the forward and backward rate constants. We have examined how the free energy of the transition state for the folding of chymotrypsin inhibitor 2 (CI2) changes with pH and temperature. In addition to the standard thermodynamic quantities, we have measured the overall acid-titration properties of the transition state and its heat capacity relative to both the denatured and native states. We were able to determine the latter by a method analogous to a well-established procedure for measuring the change in heat capacity for equilibrium unfolding: the enthalpy of activation of unfolding at different values of acid pH were plotted against the average temperature of each determination. Our results show that the transition state of CI2 has lost most of the electrostatic and van der Waals' interactions that are found in the native state, but it remains compact and this prevents water molecules from entering some parts of the hydrophobic core. The properties of the transition state of CI2 are then compared with the major folding transition state of the larger protein barnase, which folds by a multi-state mechanism, with the accumulation of a partly structured intermediate (Dphys or I). CI2 folds from a largely unstructured denatured state under physiological conditions via a transition state which is compact but relatively uniformly unstructured, with tertiary and secondary structure being formed in parallel. We term this an expanded pathway. Conversely, barnase folds from a largely structured denatured state in which elements of structure are well formed through a transition state that has islands of folded elements of structure. We term this a compact pathway. These two pathways may correspond to the two extreme ends of a continuous spectrum of protein folding mechanisms. Although the properties of the two transition states are very different, the activation barrier for folding (Dphys-->++) is very similar for both proteins.
CI2 通过简单的两态动力学作为一个单一的协同单元折叠和展开，这使得能够从正向和反向速率常数测量过渡态的性质。我们研究了胰凝乳蛋白酶抑制剂 2（CI2）折叠的过渡态自由能如何随 pH 和温度变化。除了标准的热力学量外，我们还测量了过渡态的整体酸滴定性质及其相对于变性态和天然态的热容。我们能够通过一种类似于成熟的测量平衡展开热容变化程序的方法来确定后者：绘制不同酸 pH 值下展开的活化焓与每次测定的平均温度的关系图。我们的结果表明，CI2 的过渡态已经失去了天然态中存在的大部分静电和范德华相互作用，但它仍然紧凑，这阻止了水分子进入疏水核心的某些部分。然后将 CI2 的过渡态性质与较大蛋白质巴那斯酶的主要折叠过渡态进行比较，巴那斯酶通过多态机制折叠，积累部分结构化的中间体（Dphys 或 I）。在生理条件下，CI2 从基本上无结构的变性态通过一个紧凑但相对均匀无结构的过渡态折叠，三级和二级结构并行形成。我们将此称为扩展途径。相反，巴那斯酶从一个基本上结构化的变性态折叠，其中结构元件通过一个具有折叠结构元件岛的过渡态很好地形成。我们将此称为紧凑途径。这两种途径可能对应于蛋白质折叠机制连续谱的两个极端。尽管两种过渡态的性质非常不同，但两种蛋白质折叠的活化能垒（Dphys-->++）非常相似。
