Protein Unfolding with a Steric Trap
Protein Unfolding with a Steric Trap
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DOI：
10.1021/ja905725n
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发表时间：
2009-10-07
影响因子：
15
通讯作者：
Bowie, James U.
中科院分区：
文献类型：
--
作者：
Blois, Tracy M.;Hong, Heedeok;Bowie, James U.
蛋白质折叠的研究需要一种方法来驱动解折叠，这通常是通过改变溶液条件来实现的，以有利于变性状态。这具有不期望的结果，即负责构造多肽链的分子力也改变。因此，开发可以驱动解折叠而不需要使溶剂条件不稳定的方法将是有用的。在这里，我们介绍了一种新的方法来实现这一目标，我们称之为空间捕获。在空间陷阱方法中，靶蛋白用两个空间上靠近放置的生物素标签标记，使得两个生物素标签在蛋白质展开时只能被链霉亲和素结合。因此，第二链霉亲和素的结合与靶蛋白的解折叠能量耦合。测试的模型蛋白质，二氢叶酸还原酶（DHFR）的方法，我们发现，链霉亲和素结合可以驱动展开和表观结合亲和力报告DHFR稳定性的变化。最后，通过采用野生型链霉亲和素的缓慢解离速率，我们发现DHFR可以锁定在未折叠状态。空间陷阱方法提供了一种简单的方法，用于研究蛋白质的折叠和稳定性方面的天然溶剂条件下，可用于具体展开选定的结构域，并可适用于膜蛋白。
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 goat, 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.