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。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。蛋白质在体内折叠的起始是根本不同的：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。关于新生链的生长如何影响折叠的能量格局，我们知之甚少。然而，这种差异可能有助于解释为什么一些天然状态拓扑结构在体内表现良好，但在体外作为全长多肽链重新折叠却具有挑战性。在本文中，我们将从三个独特的角度研究翻译对蛋白质折叠的影响：(1)早期事件：新生链的扩散和崩溃。我们的合作者Lisa Lapidus最近表明，相对于高浓度的变性剂，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。我们采用了Lapidus的方法来确定核糖体结合的新生链可以崩溃的程度，以及崩溃状态的分子内扩散速率，因为这些参数将影响新生链从核糖体释放后可进入的能量格局的大小和形状。(2)同义稀有密码子降低了局部翻译率，从而改变了共翻译折叠机制，可能是通过改变折叠的可达能量格局。值得注意的是，我们实验室和其他人最近的研究结果表明，对于某些蛋白质，局部翻译速率也可以改变天然结构，使其能够达到其他翻译速率模式无法达到的能量最小值。我们将测量同义密码子选择（mRNA序列）修饰蛋白质结构的能力，从而表征其对体内折叠能量景观的修饰。(3)在第一个项目期间，我们发现同义稀有密码子并非沿基因序列随机分布，而是倾向于聚集在一起，尽管这种聚集会产生负面影响（包括降低翻译率）。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（提高正确折叠蛋白质的产量）是一种影响，但更广泛的影响，包括对其他细胞功能的影响，也必须考虑在内。我们已经开发了一个可调节的体内系统，用它来回答以前无法回答的关于共翻译折叠对细胞生理学的影响的问题，包括细胞适应性。综上所述，这一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揭示了蛋白质在试管中重折叠的一般原理；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. 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. PUBLIC HEALTH RELEVANCE: Proteins are synthesized as linear strings of amino acids, but typically fold up into a three-dimensional shape in order to function. Decades of research have illuminated how full-length proteins fold in test tubes, but we still know virtually nothing about how proteins fold in the cellular environment. The proposed research will determine how the earliest cellular folding events, which occur while the protein chains are still undergoing synthesis by the ribosome, can affect the protein folding mechanism, the native protein structure and the overall fitness of the cell.