Structural and Mechanstic Aspects of Cotranslational Protein Folding

标题：共翻译蛋白质折叠的结构和机制方面这个项目将导致更好地了解蛋白质如何在细胞中获得其三维形状，为更深入地理解和控制关键的生命相关过程提供了一个途径。蛋白质的形状非常重要，因为它决定了生物活性。然而，人们对蛋白质如何在活细胞内实现其生物活性的三维形状知之甚少。这项研究将阐明蛋白质是如何从生命的最早阶段，从它们被称为核糖体的复杂机器制造的时候起，就获得精确的折叠。核糖体和分子伴侣的作用将被探讨。分子电影，显示折叠作为一个函数的时间，将产生观看微观细节的折叠过程中的真实的时间。这项研究将促进几个代表性不足的研究生的参与，并将成为学习先进的生物物理技术以及细胞中蛋白质折叠机制的基准。此外，将开发课堂演示，展示蛋白质折叠和展开过程的说明性渲染，为主动学习和本科生参与课堂提供前所未有的机会。作为该项目更广泛影响的一部分，还将组织少数民族和弱势本科生的校园预访问，以鼓励他们攻读研究生课程。尽管有大量关于纯化蛋白质在变性剂稀释和温度跳跃时折叠的文献，但关于蛋白质如何在细胞环境中折叠的信息很少，其中伴侣蛋白，分子拥挤和核糖体可以深刻地影响导致生物活性蛋白质的事件的序列和性质。该项目致力于细胞背景下折叠的最早阶段，因为蛋白质从核糖体中出现。新生链压缩和热力学稳定性的作用，以及核糖体表面的影响和全长蛋白质从核糖体释放的速率，将在分子水平上得到解决。实验将结合分子生物学和生物物理学技术进行，包括对野生型和合理设计的E. coli珠蛋白apoHmpH和其它对照新生蛋白。将在不存在和存在DnaK/DnaJ/GrpE分子伴侣的情况下收集数据，以评估这些折叠促进剂中的每一个所发挥的作用。核糖体表面和新生蛋白质之间的特异性相互作用将作为离子强度的函数进行评估。核糖体结合的新生蛋白质的释放速率将被真实的时间跟踪，以获得对伴随蛋白质诞生的事件的高分辨率见解。总的来说，这个项目将导致细胞内蛋白质折叠，基因表达的最迷人的方面之一，一个更好的理解。

TITLE: Structural and Mechanistic Aspects of Cotranslational Protein FoldingThis project will lead to a better understanding of how proteins attain their 3-dimensional shape in the cell, providing a gateway to a deeper understanding and control of crucial life-related processes. The shape of proteins is extraordinarily important because it determines biological activity. However, little is known about how proteins achieve their bioactive three-dimensional shape inside the living cell. This research will elucidate how proteins gain their precise fold from the earliest stages of their life, from the time they are made a by a complex machine known as the ribosome. The role of the ribosome and molecular helpers known as chaperones will be explored. Molecular movies, showing folding as a function of time, will be generated to watch the microscopic details of the folding process in real time. This research will foster the participation of several underrepresented graduate students, and will be a benchmark to learn advanced biophysical techniques as well as the mechanisms of protein folding in the cell. In addition, classroom demonstrations showing illustrative renderings of the protein folding and unfolding processes will be developed, providing unprecedented opportunities for active learning and for undergraduate student participation in the classroom. As part of the broader impacts of this project, campus pre-visits for minority and disadvantaged undergraduate students will also be organized, to encourage them to pursue graduate studies.Despite the extensive literature available on the folding of purified proteins upon dilution from denaturant and upon temperature-jump, there is very little information on how proteins fold in the cellular environment, where chaperones, molecular crowding and the ribosome can profoundly affect the sequence and nature of the events leading to bioactive proteins. This project addresses the earliest stages of folding in the cellular context, as proteins emerge from the ribosome. The role of nascent chain compaction and thermodynamic stability, together with the effect of the ribosomal surface and the rate of full-length protein release from the ribosome, will be addressed at the molecular level. Experiments will be carried out by a combination of molecular-biology and biophysical techniques, including fluorescence depolarization and pulsed proteolysis on wild-type and rationally designed variants of the E. coli globin apoHmpH and on other control nascent proteins. Data will be collected in the absence and presence of the DnaK/DnaJ/GrpE chaperones, to assess the role played by each of these folding facilitators. The specific interaction between the ribosomal surface and nascent proteins will be assessed as function of ionic strength. The rate of release of ribosome-bound nascent proteins will be followed in real time to gain high-resolution insights into the events accompanying protein birth. Overall, this project will lead to an improved understanding of intracellular protein folding, one of the most fascinating aspects of gene expression.