Kinetic Mechanisms of ClpA Catalyzed Polypeptide Translocation

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。本研究的目标是确定六聚体E. coli ClpA马达蛋白。 马达蛋白是各种需要机械功的细胞过程所必需的酶。因此，了解这些酶如何将ATP水解产生的能量与沿着沿着运动的能量耦合的机制，对于我们对细胞功能的基本理解是至关重要的。 研究这种马达蛋白如何运作的基本兴趣是确定马达每步在其轨道上行进多远（步长），每步使用多少能量（ATP偶联效率），它走这一步有多快（总速率和微观速率常数），以及马达保持在其轨道上与解离的概率（持续合成能力）。尽管这些蛋白质在细胞的生命周期中具有根本的重要性，但对于蛋白质解折叠酶如ClpA，描述多肽易位机制的这些参数是未知的。 确定了E.将通过采用快速混合动力学方法来实现大肠杆菌ClpA蛋白解折叠酶。 使用这些方法，本研究将产生测量的步长，耦合效率，持续合成能力，和蛋白质易位的总速率。 为了帮助解释这些参数的ClpA自缔合和核苷酸驱动的缔合过程将使用热力学和流体动力学技术进行检查。 更广泛的影响这项研究将推进跨不同领域的知识，使其他人开始检查各种多肽易位酶参与这些重要的细胞过程，如ATP依赖性蛋白水解，蛋白质聚集体的重折叠，蛋白质跨膜易位。 这项研究通过为来自不同背景的本科生、研究生和博士后研究助理提供一种工具，应用一系列生物物理方法和分子生物学技术，为发现提供了机会。 这种培训发生在研究实验室，并已转化为课堂。 首席研究员坚定地致力于扩大在科学和更广泛的研究界代表性不足的群体的参与。 这是通过在亚拉巴马和密西西比的大学和学院，包括历史上的黑人学院和大学积极寻找少数民族研究生来实现的。 此外，首席研究员是科学促进奇卡诺人和美洲原住民协会（SACNAS）的成员。 因此，首席研究员和研究小组成员将出席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。这项研究将通过保持化学系内以及整个亚拉巴马大学伯明翰校区的现有合作来加强基础设施。 该项目对社会的另一个好处是，它有可能产生在应用一系列生物物理和分子生物学技术方面训练有素的个人。 这有可能在全球范围内影响科学和技术，因为接受这些技术培训的学生将受到世界各地学术界和工业界的追捧。

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.