Blois Tracy M, Hong Heedeok, Kim Tae H, Bowie James U
Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, University of California, Los Angeles, California 90095, USA.
J Am Chem Soc. 2009 Oct 7;131(39):13914-5. doi: 10.1021/ja905725n.
The study of protein folding requires a method to drive unfolding, which is typically accomplished by altering solution conditions to favor the denatured state. This has the undesirable consequence that the molecular forces responsible for configuring the polypeptide chain are also changed. It would therefore be useful to develop methods that can drive unfolding without the need for destabilizing solvent conditions. Here we introduce a new method to accomplish this goal, which we call steric trapping. In the steric trap method, the target protein is labeled with two biotin tags placed close in space so that both biotin tags can only be bound by streptavidin when the protein unfolds. Thus, binding of the second streptavidin is energetically coupled to unfolding of the target protein. Testing the method on a model protein, dihydrofolate reductase (DHFR), we find that streptavidin binding can drive unfolding and that the apparent binding affinity reports on changes in DHFR stability. Finally, by employing the slow off-rate of wild-type streptavidin, we find that DHFR can be locked in the unfolded state. The steric trap method provides a simple method for studying aspects of protein folding and stability in native solvent conditions, could be used to specifically unfold selected domains, and could be applicable to membrane proteins.
蛋白质折叠的研究需要一种促使去折叠的方法，这通常是通过改变溶液条件以利于变性状态来实现的。这样做会产生不良后果，即负责构建多肽链的分子力也会发生变化。因此，开发无需破坏溶剂条件就能促使去折叠的方法将很有用。在此，我们介绍一种实现这一目标的新方法，我们称之为空间捕获。在空间捕获方法中，目标蛋白用两个在空间上靠近放置的生物素标签进行标记，这样只有当蛋白去折叠时两个生物素标签才能被链霉亲和素结合。因此，第二个链霉亲和素的结合在能量上与目标蛋白的去折叠相耦合。在模型蛋白二氢叶酸还原酶（DHFR）上测试该方法时，我们发现链霉亲和素结合可以促使去折叠，并且表观结合亲和力反映了DHFR稳定性的变化。最后，通过利用野生型链霉亲和素的缓慢解离速率，我们发现DHFR可以被锁定在去折叠状态。空间捕获方法为在天然溶剂条件下研究蛋白质折叠和稳定性的各个方面提供了一种简单方法，可用于特异性地使选定结构域去折叠，并且可能适用于膜蛋白。
