Native Contact Density and Nonnative Hydrophobic Effects in the Folding of Bacterial Immunity Proteins

The bacterial colicin-immunity proteins Im7 and Im9 fold by different mechanisms. Experimentally, at pH 7.0 and 10°C, Im7 folds in a three-state manner via an intermediate but Im9 folding is two-state-like. Accordingly, Im7 exhibits a chevron rollover, whereas the chevron arm for Im9 folding is linear. Here we address the biophysical basis of their different behaviors by using native-centric models with and without additional transferrable, sequence-dependent energies. The Im7 chevron rollover is not captured by either a pure native-centric model or a model augmented by nonnative hydrophobic interactions with a uniform strength irrespective of residue type. By contrast, a more realistic nonnative interaction scheme that accounts for the difference in hydrophobicity among residues leads simultaneously to a chevron rollover for Im7 and an essentially linear folding chevron arm for Im9. Hydrophobic residues identified by published experiments to be involved in nonnative interactions during Im7 folding are found to participate in the strongest nonnative contacts in this model. Thus our observations support the experimental perspective that the Im7 folding intermediate is largely underpinned by nonnative interactions involving large hydrophobics. Our simulation suggests further that nonnative effects in Im7 are facilitated by a lower local native contact density relative to that of Im9. In a one-dimensional diffusion picture of Im7 folding with a coordinate- and stability-dependent diffusion coefficient, a significant chevron rollover is consistent with a diffusion coefficient that depends strongly on native stability at the conformational position of the folding intermediate.

细菌素免疫蛋白（colicin-immunity proteins）Im7与Im9的折叠机制截然不同。在pH 7.0、10℃的实验条件下，Im7通过折叠中间体以三态路径完成折叠，而Im9的折叠则呈现类二态特征。据此，Im7表现出雪佛龙翻转（chevron rollover）现象，而Im9折叠的雪佛龙动力学臂则呈线性。
本研究借助带有/不带有额外可迁移序列依赖型能量的原生中心模型（native-centric models），探究二者折叠行为差异的生物物理基础。结果显示，单纯的原生中心模型，或是采用与残基类型无关、强度均一的非天然疏水相互作用增强的模型，均无法复现Im7的雪佛龙翻转效应。相较之下，一种更贴合实际的非天然相互作用方案——考虑残基间疏水性差异的模型——可同时复现Im7的雪佛龙翻转与Im9近乎线性的折叠雪佛龙动力学臂。
经已发表实验证实、参与Im7折叠过程中非天然相互作用的疏水残基，在本模型中被发现参与了强度最高的非天然接触。因此，本研究结果支持实验层面的观点：Im7的折叠中间体主要由涉及大型疏水残基的非天然相互作用所主导。模拟结果进一步表明，相较于Im9，Im7的局部原生接触密度更低，这一特性促进了其非天然相互作用的发生。在以坐标与稳定性依赖型扩散系数构建的Im7折叠一维扩散模型中，显著的雪佛龙翻转效应与折叠中间体构象位置处、强烈依赖于原生稳定性的扩散系数相吻合。