Influence of Translation on Protein Folding

描述(申请人提供)：成功的蛋白质折叠包括将线性多肽转化为稳定的、具有生物活性的三维结构。这一过程已经被研究了几十年，方法是在体外对纯化的全长多肽进行变性，稀释变性剂，并观察复性过程。对复性动力学和填充部分折叠构象的仔细测量揭示了许多关于复性过程的信息。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。蛋白质在体内的折叠是一个根本不同的过程：特别是，在体内折叠的起始链是一个不断增长的新生多肽链，而不是一个全长的链。关于这种根本不同的生长链格局与体外观察到的折叠机制之间的关系，我们知之甚少。然而，这种差异可能有助于解释为什么有些自然状态的拓扑在体内可以很好地表现出来，但在体外很难重新折叠。在这个方案中，翻译对体内蛋白质折叠的影响将从两个角度进行研究：(1)翻译暂停对折叠产量的影响。罕见的密码子会导致核糖体在翻译过程中暂停，文献报道将暂停与折叠产量联系在一起。对于这一建议，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停顿及其对下游折叠的影响。(2)共翻译初生链构象、体外折叠构象与游离、截短多肽构象之间的关系。蛋白质在体外重折叠能忠实地复制新生链条的构象吗？对于天然结构，多肽链的远端部分之间有接触，这是不太可能的。这些新生的链形成了哪些替代构象？它们也是由游离肽形成的吗？体外复性研究的结果揭示了蛋白质复性的一般原理；这项提议的结果将用于开发体内蛋白质折叠的原理。

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.