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。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完全不同的：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。关于新生产业链的增长如何影响折叠的能源格局，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的拓扑在体内得到很好的表现，但在体外以全长多肽链的形式进行折叠具有挑战性。在这个方案中，翻译对蛋白质折叠的影响将从三个独特的角度进行研究：(1)早期事件：新生的链扩散和崩溃。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、坍塌的蛋白质在体外的分子内扩散极其缓慢。我们采用了拉皮德斯的方法来确定与核糖体结合的初生链可以崩溃的程度，以及这种崩溃状态的分子内扩散速度，因为这些参数将影响新生链从核糖体释放后可获得的能量格局的大小和形状。(2)同义稀有密码子降低局部翻译速率，从而改变共翻译折叠机制，可能是通过改变折叠的可及能量格局。值得注意的是，我们实验室和其他实验室最近的研究结果表明，对于某些蛋白质来说，局部翻译速率也可以改变天然结构，使其能够获得在其他翻译速率模式下无法获得的最低能量。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格局的修改。(3)在第一个项目期间，我们发现同义稀有密码子并不是沿着基因序列随机分布的，而是倾向于聚集在一起，尽管这种聚集已经产生了负面影响(包括降低翻译速度)。什么积极的细胞效应可能会克服这些已知的负面影响？当然，修饰的共翻译折叠(以增加正确折叠蛋白质的产量)是一种效果，但也必须考虑更广泛的影响，包括对其他细胞功能的影响。我们已经开发了一个可调的体内系统，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，包括细胞适合性。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。几十年的体外复性研究揭示了蛋白质在试管中折叠的一般原理；这一提议的结果将用于开发蛋白质在体内折叠的原理。 与公共健康相关：蛋白质被合成为一串线性氨基酸，但为了发挥功能，蛋白质通常会折叠成三维形状。几十年的研究已经阐明了全长蛋白质如何在试管中折叠，但我们仍然对蛋白质在细胞环境中如何折叠几乎一无所知。这项拟议的研究将确定发生在蛋白质链仍在核糖体合成过程中的最早的细胞折叠事件如何影响蛋白质折叠机制、天然蛋白质结构和细胞的整体适应性。

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.