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。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。体内蛋白质折叠的起始是根本不同的：体内折叠的起始集合是生长的新生多肽链，而不是全长链。我们对新生链的增长如何影响折叠的能源格局的信息很少。然而，这种差异可能有助于解释为什么一些天然状态的拓扑结构在体内很好地代表，但在体外重新折叠为全长多肽链具有挑战性。本论文从三个方面研究翻译对蛋白质折叠的影响：（1）早期事件：新生链扩散和折叠。我们的合作者丽莎拉皮德斯最近表明，相对于高浓度的变性剂，在缓冲液中未折叠、折叠的蛋白质在体外的分子内扩散非常缓慢。我们已经调整了Lapidus的方法来确定核糖体结合的新生链可以崩溃的程度，以及该崩溃状态的分子内扩散速率，因为这些参数将影响该新生链从核糖体释放后可获得的能量景观的大小和形状。(2)同义稀有密码子降低了局部翻译速率，这可能会改变共翻译折叠机制，可能是通过改变折叠的可及能量景观。值得注意的是，我们实验室和其他实验室最近的结果表明，对于某些蛋白质，局部翻译速率也可以改变天然结构，从而获得在其他翻译速率模式下无法获得的能量最小值。我们将测量同义密码子选择（mRNA序列）修改蛋白质结构的能力，从而表征其对体内折叠能量景观的修改。(3)在第一个项目期间，我们发现同义的稀有密码子并不是沿着沿着基因序列随机分布的，而是倾向于聚集在一起，尽管这种聚集的负面影响已经确立（包括降低翻译速率）。哪些积极的细胞效应可以克服这些已知的负面影响？当然，修饰的共翻译折叠（以增加正确折叠的蛋白质的产量）是一种效应，但更广泛的效应，包括对其他细胞功能的影响，也必须考虑。我们已经开发了一种可调的体内系统，用于回答以前无法回答的关于共翻译折叠对细胞生理学（包括细胞适应性）影响的问题。总之，这项提议的结果将揭示翻译如何改变早期折叠事件，天然蛋白质结构和细胞生理学。几十年的体外重折叠研究已经揭示了蛋白质在试管中重折叠的一般原则;这项提议的结果将用于开发体内蛋白质折叠的原则。

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. What is still lacking, however, is an understanding of how these in vitro results relate to protein folding in vivo. The initiation of protein folding in vivo is fundamentally different: 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 growth of the nascent chain affects the energy landscape for folding. Yet this difference may help explain why some native state topologies are well represented in vivo, but challenging to refold in vitro as full-length polypeptide chains. In this proposal, the influence of translation on protein folding will be investigated from three unique perspectives: (1) Early events: nascent chain diffusion and collapse. Our collaborator Lisa Lapidus has recently shown that intramolecular diffusion in vitro is extremely slow for an unfolded, collapsed protein in buffer, relative to high concentrations of denaturant. We have adapted Lapidus's methods to determine the extent to which a ribosome-bound nascent chain can collapse, and the intramolecular diffusion rate for that collapsed state, as these parameters will affect the size and shape of the energy landscape accessible to that nascent chain after its release from the ribosome. (2) Synonymous rare codons reduce local translation rate, which can alter co-translational folding mechanisms, presumably by altering the accessible energy landscape for folding. Remarkably, recent results emerging from our lab and others now indicate that, for some proteins, local translation rate can also alter the native structure, enabling access to an energy minimum not accessible under other translation rate patterns. We will measure the capacity of synonymous codon selection (mRNA sequence) to modify protein structure, thereby characterizing its modifications to the energy landscape for folding in vivo. (3) In the first project period, we showed that synonymous rare codons are not randomly distributed along gene sequences, but tend to cluster together, despite established negative effects of such clustering (including reduced translation rate). What positive cellular effects might overcome these known negatives? Certainly modified co-translational folding (to increase the yield of correctly folded protein) is one effect, but broader effects, including impact on other cell functions, must also be considered. We have developed a tunable in vivo system with which to answer previously unanswerable questions regarding the effects of co-translational folding on cell physiology, including cell fitness. Taken together, results from this proposal will reveal how translation modifies early folding events, native protein structures, and cell physiology. Several decades of in vitro refolding studies have revealed general principles for protein refolding in the test tube; results from this proposal will be used to develop principles for protein folding in vivo.