RUI: Investigating Salt Induced Effects on RecA Unfolding and Nucleotide Binding

本项目的目的是确定参与核苷酸结合和离子蛋白相互作用的蛋白质结构，这些蛋白质结构导致高盐诱导的RecA活性和稳定性的变化。大肠杆菌RecA蛋白是一种依赖DNA的atp酶，在DNA修复和同源基因重组中需要形成核蛋白丝来进行DNA链交换反应。盐诱导的调节酶结构和功能的效应涉及蛋白质结构的特定变化，从而导致活性和蛋白质-蛋白质相互作用的改变。为了研究盐诱导RecA效应的来源，PI将利用差红外光谱、圆二色性和荧光来研究盐如何影响RecA的聚集和展开，同时研究盐如何调节核苷酸结合和ATP水解。差分红外光谱是一种功能强大但尚未得到充分利用的技术，它能够在单氨基酸水平上阐明多种溶液条件下核苷酸结合引起的变化。结合差异红外和圆二色性研究将使本科生获得盐诱导对稳定性和核苷酸结合的影响的补充信息。尽管几十年来对许多看似特征明确的蛋白质进行了研究，但确定参与底物结合、变构调节和离子介导过程的特定氨基酸和蛋白质结构仍然是难以捉摸的。这个项目的所有工作都将由詹姆斯麦迪逊大学的本科生完成。此外，PI在与聋哑和听力障碍学生合作方面有着悠久的历史，这些学生将被招募参与研究工作。该项目将有助于所有参与的学生获得宝贵的研究经验，并有助于招募和留住未来的科学家。此外，这些研究的结果将使我们更好地了解RecA，以及各种活性、稳定性和结构受盐存在调节的蛋白质。

The objective of this project is to identify protein structures that are involved in nucleotide binding and ion protein interactions that result in the high salt induced changes in RecA activity and stability. The Escherichia coli RecA protein is a DNA-dependent ATPase that requires formation of a nucleoprotein filament to perform the DNA strand-exchange reaction in DNA repair and homologous genetic recombination. Salt induced effects that modulate enzyme structure and function involve specific changes in protein structures that result in modified activity and protein-protein interactions. In order to investigate the origin of salt induced effects on RecA the PI will use difference infrared spectroscopy, circular dichroism and fluorescence to study how salt influences the aggregation and unfolding of RecA while simultaneously studying how salt modulates nucleotide binding and ATP hydrolysis. Difference infrared spectroscopy is a powerful yet underutilized technique that has the ability to elucidate changes induced by nucleotide-binding under a variety of solution conditions at the single amino acid level. Combining difference infrared and circular dichroism studies will enable undergraduate students to obtain complementary information about salt induced effects on stability and nucleotide binding. Despite decades of study on many seemingly well-characterized proteins, identifying the specific amino acids and protein structures that are involved in substrate binding, allosteric regulation and ion-mediated processes has continued to be elusive.All of the work in this project will be performed by undergraduates at James Madison University. In addition, the PI has an established history of working with deaf and hard-of-hearing students who will be recruited for research efforts. This project will be important in helping all the involved students gain valuable research experience and will aid in recruiting and retaining future scientists. Furthermore, the results of these studies will lead to a better understanding of not only RecA, but also the wide variety of proteins whose activity, stability and structure are modulated by the presence of salt.