Single molecule analysis of nascent protein folding

描述（由申请人提供）：本文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。我们将分析如何增加长度的纯化多肽片段的折叠，模仿新生多肽，不同于相应的全长蛋白质。然后，我们将描述单个新生多肽在翻译核糖体的背景下的折叠。这将使我们能够评估折叠能量景观如何受到蛋白质合成的矢量性质和核糖体独特环境的影响。最后，我们将研究核糖体相关的分子伴侣如何调节蛋白质折叠的自由和核糖体结合的新生链。这些发现将使我们能够绘制新生蛋白质的折叠能量景观，并深入了解支配从头蛋白质折叠的基本原理。我有一个强烈的兴趣，从头蛋白质折叠的机制和分子伴侣的调制，因为我的博士研究的开始。特别是，我已经开发了新的荧光为基础的方法来研究从头蛋白质折叠的背景下翻译的核糖体。近年来，单分子方法已成为表征本质上异质性蛋白质折叠途径的宝贵工具。Bustamante教授的实验室开发了许多利用光镊分析单分子力谱生物过程的应用。因此，他的实验室为我提供了一个无与伦比的环境，使我能够熟练地利用光镊及其在蛋白质折叠中的应用。Bustamante教授实验室的博士后学位将使我能够构建和操作光学镊子，并作为独立研究人员使用单分子方法研究从头蛋白质折叠。最终，我想建立自己的实验室，让博士生和博士后学员参与这一迷人的研究领域。 相关性：人类常见疾病，如帕金森病和阿尔茨海默病，与蛋白质错误折叠有关。对蛋白质折叠和分子伴侣功能的更透彻理解对于深入了解这些疾病状态的病理生理学至关重要，并可能最终允许开发有效的治疗干预。

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.