Kinetic Mechanisms of ClpA Catalyzed Polypeptide Translocation

ClpA催化多肽易位的动力学机制

• 批准号: 0843746
• 负责人: Aaron Lucius
• 金额: $ 54.6万
• 依托单位: University of Alabama at Birmingham
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0843746&HistoricalAwards=false
• 关键词: Kinetic; Mechanisms; ClpA; Catalyzed; Polypeptide

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).The goal of this research is to determine the mechanism of polypeptide translocation catalyzed by the hexameric E. coli ClpA motor protein. Motor proteins are essential enzymes for a variety of cellular processes that require mechanical work. Therefore, an understanding of the mechanism of how such enzymes couple the energy from ATP hydrolysis to movement along a linear track is essential to our basic understanding of cell function. Of fundamental interest in examining how such motor proteins operate is determining how far the motor travels on its track per step (step-size), how much energy is used per step (ATP coupling efficiency), how fast does it take this step (overall rate and microscopic rate-constants), and what is the probability that the motor will remain on its track vs. dissociate (processivity). Despite the fundamental importance of these proteins in the life cycle of a cell, these parameters that describe the mechanism of polypeptide translocation are not known for a protein unfoldase such as ClpA. Determination of the mechanism of protein translocation catalyzed by the E. coli ClpA protein unfoldase will be accomplished by employing rapid mixing kinetic approaches. Using these approaches, this research will yield measurements of the step-size, coupling efficiency, processivity, and overall rates for protein translocation. To aid in the interpretation of these parameters the ClpA self association and nucleotide driven association process will be examined using thermodynamic and hydrodynamic techniques. Broader Impacts This research will advance knowledge across different fields by enabling others to begin examining a variety of polypeptide translocases involved in such important cellular processes as ATP dependent proteolysis, refolding of protein aggregates, and protein translocation across membranes. This research presents opportunities for discovery by offering a vehicle for undergraduate, graduate, and postdoctoral research assistants from diverse backgrounds to apply an array of biophysical approaches and molecular biology techniques. This training occurs in the research lab and has translated into the classroom. The principal investigator is strongly committed to broadening the participation of underrepresented groups in science and the broader research community. This is being accomplished by actively seeking minority graduate students at universities and colleges across Alabama and Mississippi, including Historically Black Colleges and Universities. Additionally, the principal investigator is a member of the Society for the Advancement of Chicanos and Native Americans in Science (SACNAS). As such, the principal investigator and members of the research group will be attending the SACNAS annual national meeting in an attempt to recruit undergraduate, graduate and postdoctoral candidates from underrepresented groups. This research will enhance the infrastructure by maintaining established collaborations both within the Department of Chemistry as well as across University of Alabama at Birmingham campus. An additional benefit of this project to society is that it has the potential to yield individuals highly trained in the application of an array of biophysical and molecular biology techniques. This has the potential to globally impact science and technology because students trained in these techniques will be well sought after by both academia and industry worldwide.

该奖项根据 2009 年美国复苏和再投资法案（公法 111-5）提供资助。这项研究的目标是确定六聚体大肠杆菌 ClpA 运动蛋白催化的多肽易位机制。 运动蛋白是需要机械工作的各种细胞过程所必需的酶。因此，了解此类酶如何将 ATP 水解的能量耦合到沿线性轨道运动的机制对于我们对细胞功能的基本理解至关重要。 研究此类电机蛋白如何运作的根本兴趣在于确定电机每步在其轨道上行驶的距离（步长）、每步使用多少能量（ATP 耦合效率）、这一步的速度有多快（总体速率和微观速率常数），以及电机保持在其轨道上与解离的概率是多少（持续性）。尽管这些蛋白质在细胞生命周期中具有根本重要性，但对于 ClpA 等蛋白质解折叠酶来说，描述多肽易位机制的这些参数尚不清楚。 大肠杆菌 ClpA 蛋白解折叠酶催化的蛋白易位机制的确定将通过采用快速混合动力学方法来完成。 使用这些方法，本研究将测量蛋白质易位的步长、耦合效率、持续加工能力和总体速率。 为了帮助解释这些参数，将使用热力学和流体力学技术检查 ClpA 自关联和核苷酸驱动的关联过程。 更广泛的影响 这项研究将通过使其他人能够开始检查涉及 ATP 依赖性蛋白水解、蛋白质聚集体重折叠和蛋白质跨膜易位等重要细胞过程的各种多肽转位酶，从而推进不同领域的知识。 这项研究为来自不同背景的本科生、研究生和博士后研究助理提供了应用一系列生物物理方法和分子生物学技术的工具，从而提供了发现的机会。 这种培训在研究实验室进行，并已转化为课堂。 首席研究员坚定致力于扩大代表性不足的群体对科学和更广泛的研究界的参与。 这是通过在阿拉巴马州和密西西比州的大学和学院（包括历史上的黑人学院和大学）积极寻找少数族裔研究生来实现的。 此外，首席研究员是奇卡诺人和美洲原住民科学促进会 (SACNAS) 的成员。 因此，首席研究员和研究小组成员将参加 SACNAS 年度全国会议，试图从代表性不足的群体中招募本科生、研究生和博士后候选人。这项研究将通过维持化学系内部以及阿拉巴马大学伯明翰校区的既定合作来增强基础设施。 该项目对社会的另一个好处是，它有可能培养出经过一系列生物物理和分子生物学技术应用训练的个人。 这有可能对全球科学和技术产生影响，因为接受过这些技术培训的学生将受到全球学术界和工业界的追捧。