Effect of interdomain linker length on an antagonistic folding-unfolding equilibrium between two protein domains.
Effect of interdomain linker length on an antagonistic folding-unfolding equilibrium between two protein domains.
复制标题
DOI：
10.1016/j.jmb.2008.10.090
复制
发表时间：
2009-02-27
影响因子：
5.6
通讯作者：
Loh, Stewart N.
中科院分区：
文献类型：
--
作者：
Cutler, Thomas A.;Mills, Brandon M.;Lubin, David J.;Chong, Lillian T.;Loh, Stewart N.
一个蛋白质结构域与另一个蛋白质结构域的融合在进化和蛋白质工程实验中都是常见的事件。当插入是在一个内部位置（例如，一个表面环或转弯），而不是一个终端，构象应变可以引入到两个域。应变表现为两个区域之间的拮抗折叠展开平衡，我们之前表明可以通过耦合自由能项参数化（ΔGX）。预测应变程度主要取决于客蛋白的n - c距离与宿主蛋白表面环端之间距离的比率。在这里，我们通过将ubiquitin （Ub）插入细菌核糖核酸酶barnase （Bn）来验证这一假设，每个肽连接剂从0到10个氨基酸不等。ΔGX值是通过测量Co2+结合到Ub结构域工程位点使Bn结构域不稳定的程度来确定的。采用全原子、非强迫朗之万动力学模拟来获得机械诱导展开机制的结构洞察力。实验和计算结果表明，当连接体较长时，两个结构域在结构和能量上是不耦合的，并且ΔGX随连接体长度的减小而增大。当连接体少于两个氨基酸时，应变非常大，一个区域会展开另一个区域。然而，这种蛋白质能够重新折叠成二聚体和高阶低聚物。可能的机制是Bn畴的三维交换，减轻了构象应变。模拟表明，机械展开的有效途径始于Ub插入位点附近Bn疏水核心的破坏。
Fusion of one protein domain with another is a common event in both evolution and protein engineering experiments. When insertion is at an internal site (e.g., a surface loop or turn), as opposed to one of the termini, conformational strain can be introduced into both domains. Strain is manifested by an antagonistic folding-unfolding equilibrium between the two domains, which we previously showed can be parameterized by a coupling free-energy term (ΔGX). The extent of strain is predicted to depend primarily on the ratio of the N-to-C distance of the guest protein to the distance between ends of the surface loop in the host protein. Here, we test that hypothesis by inserting ubiquitin (Ub) into the bacterial ribonuclease barnase (Bn), using peptide linkers from zero to 10 amino acids each. ΔGX values are determined by measuring the extent to which Co2+ binding to an engineered site on the Ub domain destabilizes the Bn domain. All-atom, unforced Langevin dynamics simulations are employed to gain structural insight into the mechanism of mechanically induced unfolding. Experimental and computational results find that the two domains are structurally and energetically uncoupled when linkers are long and that ΔGX increases with decreasing linker length. When the linkers are fewer than two amino acids, strain is so great that one domain unfolds the other. However, the protein is able to refold as dimers and higher-order oligomers. The likely mechanism is a three-dimensional domain swap of the Bn domain, which relieves conformational strain. The simulations suggest that an effective route to mechanical unfolding begins with disruption of the hydrophobic core of Bn near the Ub insertion site.
登录
查看更多内容
影响因子：
2.9
作者：
Loladze, VV;Ibarra-Molero, B;Makhatadze, GI
通讯作者：
Makhatadze, GI
影响因子：
2.9
作者：
Felts, AK;Harano, Y;Levy, RM
通讯作者：
Levy, RM
影响因子：
5.6
作者：
Aroul-Selvam, R;Hubbard, T;Sasidharan, R
通讯作者：
Sasidharan, R
DOI：
10.1073/pnas.2232868100
发表时间：
2003-11-25
影响因子：
11.1
作者：
Nymeyer, H;García, AE
通讯作者：
García, AE
DOI：
10.1073/pnas.0508452103
发表时间：
2006-01-24
影响因子：
11.1
作者：
Hornak, V;Okur, A;Simmerling, C
通讯作者：
Simmerling, C