Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania.
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania.
Biophys J. 2011 Feb 2;100(3):756-764. doi: 10.1016/j.bpj.2010.12.3710.
A major challenge with testing designs of protein conformational switches is the need for experimental probes that can independently monitor their individual protein domains. One way to circumvent this issue is to use a molecular simulation approach in which each domain can be directly observed. Here we report what we believe to be the first molecular simulations of mutually exclusive folding in an engineered two-domain protein switch, providing a direct view of how folding of one protein drives unfolding of the other in a barnase-ubiquitin fusion protein. These simulations successfully capture the experimental effects of interdomain linker length and ligand binding on the extent of unfolding in the less stable domain. In addition, the effect of linker length on the potential for oligomerization, which eliminates switch activity, is in qualitative agreement with analytical ultracentrifugation experiments. We also perform what we believe to be the first study of protein unfolding via progressive localized compression. Finally, we are able to explore the kinetics of mutually exclusive folding by determining the effect of linker length on rates of unfolding and refolding of each protein domain. Our results demonstrate that molecular simulations can provide seemingly novel biological insights on the behavior of individual protein domains, thereby aiding in the rational design of bifunctional switches.
测试蛋白质构象开关设计的一个主要挑战是需要能够独立监测其各个蛋白质结构域的实验探针。一种解决此问题的方法是使用分子模拟方法，其中可以直接观察到每个结构域。在这里，我们报告了我们认为的第一个在工程双域蛋白质开关中进行互斥折叠的分子模拟，直接观察到一个蛋白质的折叠如何驱动另一个蛋白质的展开，这是在 barnase-ubiquitin 融合蛋白中实现的。这些模拟成功地捕获了实验中连接子长度和配体结合对不稳定结构域展开程度的影响。此外，连接子长度对寡聚化可能性的影响（这会消除开关活性）与分析超速离心实验定性一致。我们还进行了我们认为的首次通过渐进局部压缩的蛋白质展开研究。最后，我们能够通过确定连接子长度对每个蛋白质结构域的展开和折叠速率的影响来探索互斥折叠的动力学。我们的结果表明，分子模拟可以为单个蛋白质结构域的行为提供看似新颖的生物学见解，从而有助于理性设计双功能开关。
