Nölting B, Golbik R, Neira J L, Soler-Gonzalez A S, Schreiber G, Fersht A R
Cambridge University Chemical Laboratory and Cambridge Centre for Protein Engineering, United Kingdom.
Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):826-30. doi: 10.1073/pnas.94.3.826.
We have documented the folding pathway of the 10-kDa protein barstar from the first few microseconds at the resolution of individual residues from its well characterized denatured state. The denatured state had been shown from NMR to have flickering native-like structure in the first two of its four alpha-helices. phi-value analysis shows that the first helix becomes substantially consolidated as the intermediate is formed in a few hundred microseconds, as does the second to a lesser extent. A native-like structure then is formed in a few hundred milliseconds as the whole structure consolidates. Peptide fragments corresponding to sequences containing the first two helices separately and together as a helix-loop-helix motif have little helical structure under conditions that favor folding. The early stages of folding fit the nucleation-condensation model that was proposed for the smaller chymotrypsin inhibitor 2, which is a single module of structure and folds by two-state kinetics. The early stages of the multistate folding of the larger, multimodular, barnase have proved experimentally inaccessible. The folding pathway of barstar links those of CI2 and barnase to give a unified scheme for folding.
我们已经记录了10 kDa蛋白质巴氏星从其特征明确的变性状态开始，最初几微秒内单个残基分辨率下的折叠途径。核磁共振显示，变性状态在其四个α螺旋中的前两个螺旋中具有闪烁的类天然结构。φ值分析表明，在几百微秒内形成中间体时，第一个螺旋基本上会巩固，第二个螺旋在较小程度上也会如此。然后，随着整个结构的巩固，在几百毫秒内形成类天然结构。在有利于折叠的条件下，分别对应于包含前两个螺旋序列的肽片段以及作为螺旋-环-螺旋基序一起的肽片段几乎没有螺旋结构。折叠的早期阶段符合为较小的胰凝乳蛋白酶抑制剂2提出的成核-凝聚模型，胰凝乳蛋白酶抑制剂2是一个单一结构模块，通过两态动力学折叠。实验证明，较大的多模块核糖核酸酶Barnase的多态折叠早期阶段难以实现。巴氏星的折叠途径将胰凝乳蛋白酶抑制剂2和核糖核酸酶Barnase的折叠途径联系起来，给出了一个统一的折叠方案。
