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且具有生物活性的3D结构。这个过程已经研究了几十年，通过在体外使纯化的全长多肽变性，稀释变性剂，观察再折叠过程。对再折叠动力学和部分折叠构象的仔细测量揭示了许多关于再折叠过程的信息。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。体内蛋白质折叠是一个完全不同的过程：特别是，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。关于生长链的这种根本不同的景观如何与体外观察到的再折叠机制相关，我们知之甚少。然而，这种差异可能有助于解释为什么有些天然状态拓扑在体内表现得很好，但在体外很难重新折叠。本课题将从两个角度研究翻译对体内蛋白质折叠的影响：(1)翻译暂停对折叠率的影响。罕见的密码子可以导致核糖体在翻译过程中暂停，文献报道将暂停与折叠产率联系起来。为此，研究人员开发了新的方法来识别和调节罕见的密码子衍生的翻译停顿，以及对下游折叠的影响。(2)共翻译新生链构象、体外重折叠构象和游离截短多肽构象之间的关系。蛋白质在体外的再折叠是否忠实地再现了新生链的构象？对于天然结构，多肽链的遥远部分之间的接触，这是不可能的。这些新生链形成了什么不同的构象？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理；这一建议的结果将用于开发蛋白质在体内折叠的原理。

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.