Itzhaki L S, Otzen D E, Fersht A R
MRC Unit for Protein Function and Design Cambridge Centre for Protein Engineering, University Chemical Laboratory, U.K.
J Mol Biol. 1995 Nov 24;254(2):260-88. doi: 10.1006/jmbi.1995.0616.
The 64-residue protein chymotrypsin inhibitor 2 (CI2) is a single module of structure. It folds and unfolds as a single co-operative unit by simple two-state kinetics via a single rate determining transition state. This transition state has been characterized at the level of individual residues by analysis of the rates and equilibria of folding of some 100 mutants strategically distributed at 45 sites throughout the protein. Only one residue, a helical residue (Ala16) buried in the hydrophobic core, has its full native interaction energy in the transition state. The only region of structure which is well developed in the transition state is the alpha-helix (residues 12 to 24). But, the interactions within it are weakened, especially at the C-terminal region. The rest of the protein has varying degrees of weakly formed structure. Thus, secondary and tertiary interactions appear to form concurrently. These data, reinforced by studies on the structures of peptide fragments, fit a "nucleation-condensation" model in which the overall structure condenses around an element of structure, the nucleus, that itself consolidates during the condensation. The high energy transition state is composed of the whole of the molecule making a variety of weak interactions, the nucleus being those residues that make the strongest interactions. The nucleus here is part of the alpha-helix and some distant residues in the sequence with which it makes contacts. The remainder of the protein has to be sufficiently ordered that it provides the necessary interactions to stabilize the nucleus. The nucleus is only weakly formed in the denatured state but develops in the transition state. The onrush of stability as the nucleus consolidates its local and long range interactions is so rapid that it is not yet fully formed in the transition state. The formation of the nucleus is thus coupled with the condensation. These results are consistent with a recent simulation of the folding of a computer model protein on a lattice which is found to proceed by a nucleation-growth mechanism. We suggest that the mechanism of folding of CI2 may be a common theme in protein folding whereby fundamental folding units of larger proteins, which are modelled by the folding of CI2, form by nucleation-condensation events and coalesce, perhaps in a hierarchical manner.
由64个氨基酸残基组成的胰凝乳蛋白酶抑制剂2（CI2）是一个单一的结构模块。它通过简单的两态动力学，经由单一的速率决定过渡态，作为一个单一的协同单元进行折叠和去折叠。通过分析约100个分布在蛋白质45个位点上的策略性突变体的折叠速率和平衡，已在单个残基水平上对该过渡态进行了表征。只有一个残基，即埋在疏水核心中的螺旋残基（Ala16），在过渡态中具有其完整的天然相互作用能。在过渡态中结构发育良好的唯一区域是α螺旋（残基12至24）。但是，其中的相互作用被削弱了，尤其是在C端区域。蛋白质的其余部分具有不同程度的弱形成结构。因此，二级和三级相互作用似乎是同时形成的。这些数据，通过对肽片段结构的研究得到加强，符合“成核-凝聚”模型，在该模型中，整体结构围绕一个结构元件，即核，凝聚，而核本身在凝聚过程中巩固。高能量过渡态由整个分子进行各种弱相互作用组成，核是那些进行最强相互作用的残基。这里的核是α螺旋的一部分以及序列中与之接触的一些远处残基。蛋白质的其余部分必须足够有序，以便提供必要的相互作用来稳定核。核在变性状态下仅微弱形成，但在过渡态中发展。随着核巩固其局部和远程相互作用，稳定性的急剧增加是如此之快，以至于在过渡态中它尚未完全形成。因此，核的形成与凝聚相耦合。这些结果与最近在晶格上对计算机模型蛋白质折叠的模拟一致，该模拟发现其通过成核-生长机制进行。我们认为，CI2的折叠机制可能是蛋白质折叠中的一个共同主题，据此，由CI2的折叠所模拟的较大蛋白质的基本折叠单元通过成核-凝聚事件形成并合并，可能是以分层的方式。
