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。本研究的目的是确定六聚体大肠杆菌ClpA马达蛋白催化多肽易位的机制。运动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水解产生的能量耦合到沿着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。检查这些马达蛋白如何运作的基本兴趣是确定马达在其轨道上每一步走多远（步长），每一步使用多少能量（ATP耦合效率），它采取这一步的速度（总速率和微观速率常数），以及马达保持在其轨道上与解离（处理率）的概率是多少。尽管这些蛋白质在细胞的生命周期中具有重要的基础作用，但这些描述多肽易位机制的参数对于蛋白质展开酶（如ClpA）来说尚不为人所知。利用快速混合动力学方法确定大肠杆菌ClpA蛋白展开酶催化的蛋白易位机制。利用这些方法，本研究将测量步长、偶联效率、加工效率和蛋白质易位的总体速率。为了帮助解释这些参数，ClpA自关联和核苷酸驱动的关联过程将使用热力学和流体动力学技术进行检查。这项研究将推动不同领域的知识，使其他人能够开始研究各种多肽转位酶，这些多肽转位酶涉及ATP依赖的蛋白质水解、蛋白质聚集体的重折叠和蛋白质跨膜易位等重要的细胞过程。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一个应用一系列生物物理方法和分子生物学技术的工具，为发现提供了机会。这种训练在研究实验室进行，并已转化为课堂。首席研究员坚定地致力于扩大代表性不足的群体在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积极寻找少数民族研究生来实现的，包括历史上的黑人学院和大学。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之间的既定合作来加强基础设施。这个项目对社会的另一个好处是，它有可能培养出在应用一系列生物物理和分子生物学技术方面受过高度训练的个人。这有可能在全球范围内影响科学和技术，因为在这些技术方面受过培训的学生将受到全世界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.