Single molecule analysis of nascent protein folding

描述(申请人提供)：这里提出的实验将在越来越复杂和生物相关的背景下分析新生蛋白质的折叠。我们将分析增加长度的纯化多肽片段的折叠与相应全长蛋白质的折叠有何不同。然后，我们将在翻译核糖体的背景下描述单个新生多肽的折叠。这将使我们能够评估折叠能量格局如何受到蛋白质合成的矢量性质和核糖体的独特环境的影响。最后，我们将研究核糖体相关的伴侣如何调节自由和核糖体结合的新生链蛋白折叠。这些发现将使我们能够绘制新生蛋白质的折叠能量图景，并提供对支配从头蛋白质折叠的基本原理的洞察。自从我开始攻读博士学位以来，我就对从头蛋白折叠的机制以及分子伴侣对其调节产生了浓厚的兴趣。特别是，我开发了新的基于荧光的方法来研究核糖体翻译背景下的从头蛋白质折叠。近年来，单分子方法已经成为表征本质上不同的蛋白质折叠途径的宝贵工具。许多利用光镊单分子力谱分析生物过程的应用都是在布斯塔曼特教授的实验室里开发出来的。因此，他的实验室为我提供了一个无与伦比的环境，让我精通光学镊子的使用及其在蛋白质折叠中的应用。布斯塔曼特教授实验室的博士后学位将使我能够建造和操作光学镊子，并作为独立研究员使用单分子方法研究从头开始的蛋白质折叠。最终，我想建立自己的实验室，让博士生和博士后实习生参与这一迷人的研究领域。 相关性：普遍存在的人类疾病，如帕金森氏症和阿尔茨海默氏症，与蛋白质错误折叠有关。更彻底地了解蛋白质折叠和分子伴侣的功能对于深入了解这些疾病状态背后的病理生理机制是必不可少的，并最终可能允许开发有效的治疗干预措施。

DESCRIPTION (provided by applicant): The experiments proposed here will analyze the folding of nascent proteins in an increasingly more complex and biologically relevant context. We will analyze how the folding of purified polypeptide fragments of increasing length, mimicking nascent polypeptides, differs from that of the corresponding full length proteins. Then, we will characterize the folding of individual nascent polypeptides in the context of translating ribosomes. This will allow us to assess how the folding energy landscape is influenced by the vectorial nature of protein synthesis and the unique environment of the ribosome. Finally, we will investigate how ribosome-associated chaperones modulate protein folding of both free and ribosome-bound nascent chains. These findings will allow us to map the folding energy landscapes of nascent proteins and provide insight into the fundamental principles that govern de novo protein folding. I have had a strong interest in the mechanism of de novo protein folding and its modulation by molecular chaperones since the beginning of my doctoral studies. Particularly, I have developed novel fluorescence-based methodologies to investigate de novo protein folding in the context of translation by the ribosome. In recent years, single molecule methodologies have become invaluable tools for the characterization of intrinsically heterogeneous protein folding pathways. Many of the applications that utilize optical tweezers to analyze biological processes with single molecule force spectroscopy have been developed in the laboratory of Prof. Bustamante. Thus, his laboratory provides an unequaled environment for me to become proficient in the utilization of optical tweezers and their application to protein folding. A postdoctorate in Prof. Bustamante's laboratory will enable me to build and operate optical tweezers and to study de novo protein folding with single molecule methods as an independent researcher. Ultimately, I would like to establish my own laboratory to engage PhD students and postdoctoral trainees in this fascinating area of research. RELEVANCE: Prevalent human disorders, such as Parkinson's and Alzheimer's diseases, are associated with protein misfolding. A more thorough understanding of protein folding and the function of molecular chaperones is imperative to gain insight into the pathophysiology underlying these disease states and may ultimately allow the development of effective therapeutic intervention.