Least activation path for protein folding: investigation of staphylococcal nuclease folding by stopped-flow circular dichroism.

Is the pathway of protein folding determined by the relative stability of folding intermediates, or by the relative height of the activation barriers leading to these intermediates? This is a fundamental question for resolving the Levinthal paradox, which stated that protein folding by a random search mechanism would require a time too long to be plausible. To answer this question, we have studied the guanidinium chloride (GdmCl)-induced folding/unfolding of staphylococcal nuclease [(SNase, formerly EC 3.1.4.7; now called microbial nuclease or endonuclease, EC 3.1.31.1] by stopped-flow circular dichroism (CD) and differential scanning microcalorimetry (DSC). The data show that while the equilibrium transition is a quasi-two-state process, kinetics in the 2-ms to 500-s time range are triphasic. Data support the sequential mechanism for SNase folding: U3 <--> U2 <--> U1 <--> N0, where U1, U2, and U3 are substates of the unfolded protein and N0 is the native state. Analysis of the relative population of the U1, U2, and U3 species in 2.0 M GdmCl gives delta-G values for the U3 --> U2 reaction of +0.1 kcal/mol and for the U2 --> U1 reaction of -0.49 kcal/mol. The delta-G value for the U1 --> N0 reaction is calculated to be -4.5 kcal/mol from DSC data. The activation energy, enthalpy, and entropy for each kinetic step are also determined. These results allow us to make the following four conclusions. (i) Although the U1, U2, and U3 states are nearly isoenergetic, no random walk occurs among them during the folding. The pathway of folding is unique and sequential. In other words, the relative stability of the folding intermediates does not dictate the folding pathway. Instead, the folding is a descent toward the global free-energy minimum of the native state via the least activation path in the vast energy landscape. Barrier avoidance leads the way, and barrier height limits the rate. Thus, the Levinthal paradox is not applicable to the protein-folding problem. (ii) The main folding reaction (U1 --> N0), in which the peptide chain acquires most of its free energy (via van der Waals' contacts, hydrogen bonding, and electrostatic interactions), is a highly concerted process. These energy-acquiring events take place in a single kinetic phase. (iii) U1 appears to be a compact unfolded species; the rate of conversion of U2 to U1 depends on the viscosity of solution. (iv) All four relaxation times reported here depend on GdmCl concentrations: it is likely that none involve the cis/trans isomerization of prolines. Finally, a mechanism is presented in which formation of sheet-like chain conformations and a hydrophobic condensation event precede the main-chain folding reaction. IMAGES:

蛋白质折叠的路径究竟是由折叠中间体的相对稳定性决定，还是由通向这些中间体的活化能垒的相对高度所支配？这是解析莱文塔尔悖论（Levinthal paradox）的核心问题——该悖论指出，若通过随机搜索机制进行蛋白质折叠，所需时间将长得不符合实际。

为解答这一问题，我们借助停流圆二色光谱（stopped-flow circular dichroism, CD）与差示扫描微量热法（differential scanning microcalorimetry, DSC），研究了氯化胍（guanidinium chloride, GdmCl）诱导的葡萄球菌核酸酶（staphylococcal nuclease, SNase，原EC 3.1.4.7；现称为微生物核酸酶或核酸内切酶，EC 3.1.31.1）的折叠/解折叠过程。

实验数据表明，尽管平衡转变为准两态过程，但2毫秒至500秒时间范围内的动力学过程呈现三相特征。数据支持葡萄球菌核酸酶折叠的序贯机制：U3 ↔ U2 ↔ U1 ↔ N0，其中U1、U2、U3为未折叠蛋白的亚状态，N0为天然状态。

对2.0 M GdmCl中U1、U2、U3三种物种的相对占比进行分析后，得到U3→U2反应的ΔG值为+0.1 kcal/mol，U2→U1反应的ΔG值为-0.49 kcal/mol。结合DSC数据，可计算得到U1→N0反应的ΔG值为-4.5 kcal/mol。此外，我们还测定了各动力学步骤的活化能、焓与熵。

基于上述结果，我们可得出以下四点结论：
（i）尽管U1、U2、U3三种状态的能量近乎相等，但折叠过程中并未在它们之间发生随机游走。折叠路径具有唯一性且遵循序贯模式，换言之，折叠中间体的相对稳定性并非折叠路径的决定因素。相反，在广阔的能量景观中，折叠是通过活化能最低的路径向天然状态的全局自由能极小值趋近的过程：规避能垒主导了折叠方向，而能垒高度则限制了折叠速率。因此，莱文塔尔悖论并不适用于该蛋白质折叠问题。
（ii）主折叠反应（U1→N0）是一个高度协同的过程——在此过程中，肽链通过范德华接触、氢键与静电相互作用获得大部分自由能，这些能量获取事件均发生在单一动力学阶段。
（iii）U1似乎是一种紧凑的未折叠物种；U2向U1的转化速率依赖于溶液的黏度。
（iv）本文报道的所有四种弛豫时间均与GdmCl浓度相关，这提示其中并不涉及脯氨酸的顺/反异构化过程。

最后，我们提出了一种折叠机制：在主链折叠反应发生前，先形成片状链构象并伴随疏水凝聚事件。
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