Otzen Daniel E, Oliveberg Mikael
Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, Aalborg, DK-9000, Denmark.
J Mol Biol. 2002 Apr 5;317(4):613-27. doi: 10.1006/jmbi.2002.5423.
An increasing number of folding studies of two-state proteins shows that point mutations sometimes change the kinetic m-values, leading to kinks and curves in the chevron plots. The molecular origin of these changes is yet unclear although it is speculated that they are linked to structural rearrangement of the transition state or to accumulation of meta-stable intermediates. To shed more light on this issue, we present here a combined m and phi-value analysis of the split beta-alpha-beta protein S6. Wild-type S6 displays classical two-state kinetics with v-shaped chevron plot, but a majority of its mutants display distinct m-value changes or curved chevrons. We observe that this kinetic aberration of S6 is linked to mutations that are clustered in distinct regions of the native structure. The most pronounced changes, i.e. decrease in the m-value for the unfolding rate constant, are seen upon truncation of interactions between the N and C termini, whereas mutations in the centre of the hydrophobic core show smaller or even opposed effects. As a consequence, the calculated phi-values display a systematic increase upon addition of denaturant. In the case of S6, the phenomenon seems to arise from a general plasticity of the different species on the folding pathway. That is, the structure of the denatured ensemble, the transition state, and the native ground-state for unfolding seem to change upon mutation. From these changes, it is concluded that interactions spanning the centre of the hydrophobic core form early in folding, whereas the entropically disfavoured interactions linking the N and C termini consolidate very late, mainly on the down-hill-side of the folding barrier.
越来越多对两态蛋白质的折叠研究表明，点突变有时会改变动力学m值，导致在V形曲线图谱中出现扭结和曲线。尽管据推测这些变化与过渡态的结构重排或亚稳态中间体的积累有关，但其分子起源尚不清楚。为了更清楚地了解这个问题，我们在此展示了对分裂β-α-β蛋白S6的m值和φ值的联合分析。野生型S6呈现出具有V形曲线图谱的经典两态动力学，但它的大多数突变体表现出明显的m值变化或弯曲的V形曲线。我们观察到S6的这种动力学异常与聚集在天然结构不同区域的突变有关。最显著的变化，即解折叠速率常数的m值降低，在N端和C端之间的相互作用被截断时出现，而疏水核心中心的突变显示出较小甚至相反的影响。因此，在添加变性剂时，计算出的φ值呈现出系统性的增加。对于S6来说，这种现象似乎源于折叠途径上不同物种的一般可塑性。也就是说，变性态系综、过渡态以及解折叠的天然基态的结构似乎在突变时发生了变化。从这些变化可以得出结论，跨越疏水核心中心的相互作用在折叠早期形成，而连接N端和C端的熵不利相互作用很晚才巩固，主要是在折叠屏障的下坡侧。
