CAREER: Elucidating the role of ATP in Cytosolic Iron Sulfur Cluster Biogenesis

波士顿大学的Deborah Perlstein教授获得了生命过程化学项目的终身成就奖，她正在研究真核细胞的细胞质中铁和硫的簇是如何组装并转移到合适的蛋白质上的。簇生物合成对细胞生长和分裂至关重要，因为需要铁硫簇的蛋白质在DNA复制和修复以及许多其他重要的细胞功能中起着核心作用。尽管它的重要性毋庸置疑，但科学家们对这些铁硫簇如何合成并最终转化为适当的蛋白质/酶的分子细节知之甚少。Perlstein教授的实验室正在结合遗传学、生物化学和生物物理学的方法来阐明蛋白质支架上的簇是如何组装的，以及最终将簇插入蛋白质所需的后续步骤。该研究为本科生和研究生以及博士后提供了良好的培训环境，使他们具备成为化学-生物学领域研究领导者所需的专业知识。此外，通过开发与研究计划密切相关的本科生物化学讲座和实验课程以及K-12 STEM外展计划的课程，Perlstein教授的工作拓宽了对生物化学和生物技术研究的兴趣，这构成了波士顿地区就业机会的重要组成部分。该项目的具体目标是揭示为什么酵母胞质铁硫簇脚手架蛋白需要ATP水解才能发挥活性。由于酵母酶及其在Mrp/Nbp35家族中的同系物在P-loop NTPases的异常Walker a家族中形成了一个独特的亚家族，Perlstein博士和她的团队假设ATP水解被用来诱导支架内的构象变化，用于协调支架上新生簇的组装和该簇向受体的转移。这种核苷酸驱动的开关可以通过簇支架位点和atp酶位点之间的直接变构通信，通过调节与其他簇生物发生蛋白的动态相互作用，或通过这两种机制的组合，影响簇组装和/或支架的传递功能。通过确定ATP酶突变体如何在体外影响簇支架活性，确定与支架相互作用的其他因素，并阐明ATP水解的动力学机制如何受到支架簇或其他簇组装因素的影响，研究人员希望揭示这个ATP水解铁硫簇支架大家族如何利用核苷酸水解来协调支架内新生簇的组装，以及随后将簇从支架动员到目标所需的运输步骤。这项研究与一项教育计划相结合，旨在通过与波士顿大学学习资源网络的合作，使K-12学生接触化学-生物学界面的研究和就业机会。此外，Perlstein教授还为计划从事研究工作的学生量身定制了一门针对本科化学和生物学专业的强化生物化学课程。

With this CAREER award from the Chemistry of Life Processes Program, Professor Deborah Perlstein at Boston University is investigating how clusters of iron and sulfur are assembled and transferred to their appropriate proteins in the cytosol of eukaryotic cells. Cluster biosynthesis is essential for cell growth and division since proteins requiring iron sulfur clusters play central roles in DNA replication and repair as well as many other important cellular functions. Despite its unquestionable importance, scientists understand remarkably little about the molecular details of how these iron sulfur clusters are synthesized and ultimately transferred into the appropriate proteins/enzymes. Professor Perlstein's lab is using a combination of genetic, biochemical, and biophysical approaches to shed light on how assembly of a cluster on a protein scaffold takes place as well as on the subsequent steps required to ultimately insert the cluster into a protein. The research provides an excellent training environment for students at the undergraduate and graduate students as well as postdoctoral fellows, equipping them with the expertise required to become leaders in research at the chemistry-biology interface. Furthermore, by developing the curriculum for an undergraduate biochemistry lecture and laboratory course and a K-12 STEM outreach program both intimately connected with the research plan, Professor Perlstein's work broadens interest in biochemistry and biotechnology research which forms a significant portion of the job opportunities in the Boston area. The specific goal of this project is to uncover why the yeast cytosolic iron sulfur cluster scaffolding proteins require ATP hydrolysis for their activity. Since the yeast enzyme and its homologs in the Mrp/Nbp35 family of cluster scaffolding ATPases form a distinct subfamily within the deviant Walker A family of P-loop NTPases, Dr. Perlstein and her group hypothesize that ATP hydrolysis is utilized to induce a conformational change within the scaffold that is used to coordinate assembly of a nascent cluster on the scaffold with the transfer of this cluster to a recipient. This nucleotide driven switch could impact cluster assembly and/or transfer functions of the scaffold via direct allosteric communication between the cluster scaffolding site and the ATPase site, via regulation of dynamic interactions with other cluster biogenesis proteins, or via a combination of these two mechanisms. By determining how ATPase mutants affect cluster scaffolding activities in vitro, identifying other factors that interact with the scaffold, and elucidating how kinetic mechanism of ATP hydrolysis is affected by scaffolded cluster or other cluster assembly factors, the researchers expect to uncover how nucleotide hydrolysis is exploited by this large family of ATP hydrolyzing iron sulfur cluster scaffolds to coordinate assembly of a nascent cluster within the scaffolding site with the subsequent trafficking steps required to mobilize the cluster from scaffold to target. This research is integrated with an educational plan aimed at exposing K-12 students to research and career opportunities at the chemistry-biology interface via collaboration with Boston University's Learning Resource Network. Furthermore, Professor Perlstein leads the development of an Intensive Biochemistry course for undergraduate chemistry and biology majors tailored to the needs of students planning to pursue research careers.