Liu Kaixian, Rehfus Joseph E, Mattson Elliot, Kaiser Christian M
Department of Biology, Johns Hopkins University, Baltimore, Maryland.
Program in Cell, Molecular, Developmental Biology, and Biophysics, Johns Hopkins University, Baltimore, Maryland.
Protein Sci. 2017 Jul;26(7):1439-1451. doi: 10.1002/pro.3189. Epub 2017 May 19.
Correct folding is a prerequisite for the biological activity of most proteins. Folding has largely been studied using in vitro refolding assays with isolated small, robustly folding proteins. A substantial fraction of all cellular proteomes is composed of multidomain proteins that are often not amenable to this approach, and their folding remains poorly understood. These large proteins likely begin to fold during their synthesis by the ribosome, a large molecular machine that translates the genetic code. The ribosome affects how folding proceeds, but the underlying mechanisms remain largely obscure. We have utilized optical tweezers to study the folding of elongation factor G, a multidomain protein composed of five domains. We find that interactions among unfolded domains interfere with productive folding in the full-length protein. The N-terminal G-domain constitutes an independently folding unit that, upon in vitro refolding, adopts two similar states that correspond to the natively folded and a non-native, possibly misfolded structure. The ribosome destabilizes both of these states, suggesting a mechanism by which terminal misfolding into highly stable, non-native structures is avoided. The ribosome may thus directly contribute to efficient folding by modulating the folding of nascent multidomain proteins.
正确折叠是大多数蛋白质具有生物活性的前提条件。折叠过程主要是通过对分离出的、易于折叠的小蛋白质进行体外重折叠实验来研究的。所有细胞蛋白质组中有很大一部分是由多结构域蛋白质组成的，这些蛋白质通常不适用于这种方法，其折叠过程仍知之甚少。这些大蛋白质可能在核糖体（一种翻译遗传密码的大型分子机器）合成它们的过程中就开始折叠了。核糖体影响折叠的进行方式，但其潜在机制仍基本不明。我们利用光镊研究了延伸因子G（一种由五个结构域组成的多结构域蛋白质）的折叠过程。我们发现未折叠结构域之间的相互作用会干扰全长蛋白质的有效折叠。N端G结构域构成一个独立的折叠单元，在体外重折叠时会呈现出两种相似的状态，分别对应天然折叠结构和非天然、可能错误折叠的结构。核糖体使这两种状态都不稳定，这表明存在一种避免末端错误折叠成高度稳定的非天然结构的机制。因此，核糖体可能通过调节新生多结构域蛋白质的折叠直接促进有效折叠。
