López-Hernández E, Serrano L
EMBL, Heidelberg, Germany.
Fold Des. 1996;1(1):43-55.
Protein engineering analysis has been used as a tool to determine the structure of the transition state of two different proteins: CI-2 and barnase. CI-2 belongs to the group of small, globular proteins with no disulphide bonds that fold via a two-state mechanism. Barnase is a larger protein (110 aa) and displays a folding intermediate. The structure of the transition state of both proteins is quite different. Whereas in CI-2 no region is fully native and it looks like an expanded form of the folded state, in barnase several regions are folded and the rate-limiting step seems to be the consolidation of the hydrophobic core. On the basis of these results, a unified scheme for the transition state of protein folding has been presented. We decided to characterize the folding pathway, or pathways, present in the alpha/beta parallel family of proteins using one of the smallest members, CheY (129 aa), as a model case.
The folding pathway of CheY contains, as does that of barnase, a kinetic intermediate. The picture obtained for CheY from the equilibrium and kinetic analyses of several mutations scattered throughout the whole protein is different from that found for barnase. On the basis of the experimental results and the structure of CheY, the protein can be divided into two subdomains (from beta-strand 1 to beta-strand 3 and from beta-strand 3 to the C terminus). Whereas the structure of the first subdomain in the transition state resembles that found for the CI-2 protein, the second subdomain is compact but unstructured. The packing of the first alpha-helix against beta-strands 1 and 2 seems to be the nucleus around which the rest of the protein folds.
Comparison of the transition state of barnase with those of CheY and CI-2 indicates that different proteins have different transition states, probably depending on the energetics and the position of the rate-limiting step in the folding pathway. CheY appears to fold through a nucleation/condensation mechanism as has been found for CI-2. The rate-determining step in some multimodular proteins could be the formation of a stable domain, with the less stable domains folding after the major rate-determining step.
蛋白质工程分析已被用作一种工具，以确定两种不同蛋白质（CI-2和巴纳酶）过渡态的结构。CI-2属于无二硫键的小的球状蛋白质组，通过两态机制折叠。巴纳酶是一种较大的蛋白质（110个氨基酸），具有折叠中间体。两种蛋白质过渡态的结构有很大差异。在CI-2中，没有区域是完全天然的，它看起来像是折叠态的扩展形式，而在巴纳酶中，几个区域是折叠的，限速步骤似乎是疏水核心的巩固。基于这些结果，提出了蛋白质折叠过渡态的统一方案。我们决定以α/β平行蛋白家族中最小的成员之一CheY（129个氨基酸）为模型，来表征该家族中存在的折叠途径。
CheY的折叠途径与巴纳酶一样，包含一个动力学中间体。通过对分布在整个蛋白质中的几个突变进行平衡和动力学分析得到的CheY的情况，与巴纳酶不同。基于实验结果和CheY的结构，该蛋白质可分为两个亚结构域（从β链1到β链3以及从β链3到C端）。在过渡态中，第一个亚结构域的结构类似于在CI-2蛋白中发现的结构，而第二个亚结构域是紧密的但未结构化。第一个α螺旋与β链1和2的堆积似乎是蛋白质其余部分围绕其折叠的核心。
将巴纳酶的过渡态与CheY和CI-2的过渡态进行比较表明，不同的蛋白质具有不同的过渡态，这可能取决于折叠途径中的能量学和限速步骤的位置。正如在CI-2中发现的那样，CheY似乎通过成核/凝聚机制折叠。一些多模块蛋白质中的限速步骤可能是稳定结构域的形成，较不稳定的结构域在主要限速步骤之后折叠。
