Friedel Miriam, Baumketner Andrij, Shea Joan-Emma
Department of Physics, University of California, Santa Barbara, CA 93106, USA.
Proc Natl Acad Sci U S A. 2006 May 30;103(22):8396-401. doi: 10.1073/pnas.0601210103. Epub 2006 May 18.
The folding mechanisms of proteins are increasingly being probed through single-molecule experiments in which the protein is immobilized on a surface. Nevertheless, a clear understanding of how the surface might affect folding, and whether or not it changes folding from its bulk behavior, is lacking. In this work, we use molecular dynamics simulations of a model beta-barrel protein tethered to a surface to systematically investigate how the surface impacts folding. In the bulk, this protein folds in a three-state manner through a compact intermediate state, and its transition state (TS) has a well formed hydrophobic core. Upon tethering, we find that folding rates and stability are impacted differently by the surface, with dependencies on both the length and location of the tether. Significant changes in folding times are observed for tether points that do not alter the folding temperature. Tethering also locally enhances the formation of structure for residues proximal to the tether point. We find that neither the folding mechanism nor the TS of this protein are altered if the tether is in a fully structured or completely unstructured region of the TS. By contrast, tethering in a partially structured region of the TS leads to dramatic changes. For one such tether point, the intermediate present in bulk folding is eliminated, leading to a two-state folding process with a heterogeneous, highly unstructured TS ensemble. These results have implications for both the design of single-molecule experiments and biotechnological applications of tethered proteins.
蛋白质的折叠机制越来越多地通过单分子实验来探究，在这些实验中，蛋白质被固定在一个表面上。然而，目前仍缺乏对表面如何影响折叠以及它是否会改变蛋白质在本体状态下的折叠行为的清晰理解。在这项工作中，我们使用分子动力学模拟研究了一个与表面相连的β桶状模型蛋白，以系统地探究表面如何影响折叠。在本体状态下，这种蛋白质通过一个紧密的中间态以三态方式折叠，其过渡态（TS）具有一个形成良好的疏水核心。连接到表面后，我们发现折叠速率和稳定性受到表面的不同影响，这取决于连接链的长度和位置。对于那些不改变折叠温度的连接点，折叠时间会有显著变化。连接还会局部增强连接点附近残基的结构形成。我们发现，如果连接链位于过渡态的完全结构化或完全非结构化区域，该蛋白质的折叠机制和过渡态都不会改变。相比之下，在过渡态的部分结构化区域进行连接会导致显著变化。对于这样一个连接点，本体折叠中存在的中间态被消除，导致一个具有异质、高度非结构化过渡态集合的两态折叠过程。这些结果对单分子实验的设计以及连接蛋白的生物技术应用都有启示。
