Influence of Translation on Protein Folding

描述（由申请人提供）：成功的蛋白质折叠涉及将线性多肽转化为稳定的生物活性3D结构。这个过程已经研究了几十年，通过在体外变性纯化的全长多肽，稀释变性剂，并观察重折叠过程。重折叠动力学和填充部分折叠构象的仔细测量揭示了很多关于重折叠过程。然而，仍然缺乏的是对这些体外结果如何与体内蛋白质折叠相关的理解。蛋白质在体内的折叠是一个根本不同的过程：特别是，在体内折叠的起始集合是一个正在生长的新生多肽链，而不是全长链。对于这种生长链的根本不同景观与体外观察到的重折叠机制之间的关系，我们几乎没有什么信息。然而，这种差异可能有助于解释为什么有一些天然状态的拓扑结构，在体内表现良好，但难以在体外重折叠。本论文从两个方面研究了翻译对蛋白质折叠的影响：（1）翻译停顿对蛋白质折叠产率的影响。罕见的密码子可以导致核糖体在翻译过程中暂停，并且文献报道将暂停与折叠产量联系起来。对于这一提议，已经开发了新的方法来识别和调节罕见的密码子衍生的翻译暂停，以及对下游折叠的影响。(2)共翻译新生链构象、体外重折叠构象与游离截短多肽构象之间的关系。体外蛋白质重折叠是否忠实地再现了新生链的构象？对于天然结构，多肽链的远端部分之间有接触，这是不可能的。这些新生的链形成了什么样的构象？它们也是由游离肽形成的吗？体外重折叠研究的结果揭示了蛋白质重折叠的一般原则，从这一建议的结果将被用来制定原则，在体内蛋白质折叠。

DESCRIPTION (provided by applicant): Successful protein folding involves the conversion of a linear polypeptide into a stable and biologically active 3D structure. This process has been studied for decades by denaturing purified, full-length polypeptides in vitro, diluting away the denaturant, and observing the refolding process. Careful measurements of refolding kinetics and populated partially folded conformations have revealed much about the refolding process. What is still lacking, however, is an understanding of how these in vitro results relate to protein folding in vivo. Protein folding in vivo is a fundamentally different process: In particular, the starting ensemble for folding in vivo is a growing nascent polypeptide chain, rather than a full-length chain. We have very little information about how this fundamentally different landscape for the growing chain relates to refolding mechanisms observed in vitro. Yet this difference may help explain why there are some native state topologies that are well represented in vivo, but difficult to refold in vitro. In this proposal, the influence of translation on protein folding in vivo will be studied from two perspectives: (1) The effects of translation pausing on folding yield. Rare codons can cause the ribosome to pause during translation, and literature reports have connected pauses with folding yield. For this proposal, novel methods have been developed to identify and modulate rare codon-derived translation pauses, and the effects on downstream folding. (2) The relationships between co-translational nascent chain conformations, in vitro refolding conformations, and the conformations of free, truncated polypeptides. Does protein refolding in vitro faithfully reproduce the conformations of nascent chains? For native structures, with contacts between distant portions of the polypeptide chain, this is unlikely. What alternative conformations are formed by these nascent chains? Are they also formed by free peptides? Results from in vitro refolding studies have revealed general principles for protein refolding; results from this proposal will be used to develop principles for protein folding in vivo.