CAREER: Structural and Mechanistic Studies on a Iron-Sulfur Cluster-Based Nitric Oxide Sensor

由于铁(Fe)和硫(S)离子都是高度活性的，含有多个铁和硫组合(Fe-S簇)的蛋白质可以在细菌如何感知和快速适应环境压力方面发挥多种作用。铁-S簇蛋白的适当功能是细菌适应能力所必需的。这些蛋白质通常赋予对宿主致病的细菌持久性和抗生素耐药性。尽管这些铁-S簇蛋白很重要，但由于使用这些氧敏感蛋白的技术挑战，对它们在细菌中的功能及其潜在作用机制的知识存在严重空白。生命过程化学和既定的激励竞争性研究计划(EPSCoR)联合资助了内布拉斯加州大学林肯分校的张丽梅博士。张教授建立了一个综合的研究和教育项目，其目标是破译放线杆菌中一种非常重要但尚未得到充分研究的铁-S簇蛋白WhiB1的独特作用模式的结构基础。从这个项目中获得的知识可能有助于对抗致病放线菌，如致命的、持久的结核分枝杆菌。这项研究还可能改进利用放线菌生产生物活性分子，这反过来可能对农业有利。这项研究纳入了一系列旨在招募和培训下一代STEM研究人员的教育和外联活动，重点是在科学界代表性不足的群体。它还通过与包括K-12、本科生和研究生以及普通公众在内的受众接触来促进科学素养。WhiB1是一种基于[4Fe-4S]簇的一氧化氮(NO)传感器，广泛存在于放线杆菌中，在结核分枝杆菌(Mtb)中是必不可少的。WhiB1与Mtb的无应激反应和休眠的启动有关。尽管对该蛋白进行了数十年的研究，但在确定WhiB1的作用模式方面仍然存在关键的知识空白，因为它在支持活跃的细胞生长以及在有氧条件下选择性地感知和调节基因表达以响应细胞的NO水平方面发挥着重要作用。除了NO感觉和应激反应的细节外，WhiB1代表了一个参与信号传递的未被充分研究的单体转录因子大家族，其潜在的作用机制尚不清楚。在这个项目中，张博士和她的研究小组使用Mtb WhiB1作为模型蛋白质和一套高端结构、光谱和分子工具，以促进对[4Fe-4S]簇依赖的蛋白质折叠和动力学控制、[4Fe-4S]簇转录因子的非选择性以及WhiB1类单体转录因子的作用模式的机械理解。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Because iron (Fe) and sulfur (S) ions are both highly reactive, proteins that contain multi-iron and sulfur assemblies (Fe-S clusters), can play versatile roles in how bacteria sense and rapidly adapt to environmental stress. Proper function of Fe-S cluster proteins is required for the fitness of bacteria. These proteins often confer persistence and antibiotic resistance to bacteria that are pathogenic to their host. Despite the importance of these Fe-S cluster proteins, there are critical gaps in knowledge about how they function in bacteria and their underlying mechanisms of action due to the technical challenges of working with these oxygen-sensitive proteins. The Chemistry of Life Processes and Established Program to Stimulate Competitive Research (EPSCoR) Programs jointly fund Dr. Limei Zhang at the University of Nebraska-Lincoln. Professor Zhang builds an integrated research and education program whose goal is to decipher the structural basis for the unique mode of action of a very important, but understudied, Fe-S cluster protein in Actinobacteria called WhiB1. The knowledge gained from this project may help combat pathogenic actinobacteria such as the deadly, persistent pathogen Mycobacterium tuberculosis. The research may also improve production of bioactive molecules using actinobacteria that could, in turn, have benefits for the agriculture. This research is integrated into a series of education and outreach activities designed to recruit and train next-generation STEM researchers, with an emphasis on groups underrepresented in science. It also promotes science literacy by engaging with audiences that include K-12, undergraduate, and graduate students, as well as the general public. WhiB1 is a [4Fe-4S] cluster-based nitric oxide (NO) sensor widely distributed in actinobacteria and essential in Mycobacterium tuberculosis (Mtb). WhiB1 is linked to the NO stress response and initiation of dormancy in Mtb. Despite decades of investigation on this protein, crucial knowledge gaps remain in defining the mode of action of WhiB1 for its essential role in supporting active cell growth and for selectively sensing and regulating gene expression in response to cellular NO levels under aerobic conditions. Beyond the details about NO sensing and stress response, WhiB1 represents a large family of understudied monomeric transcription factors involved in signaling, of which the underlying mechanisms of action are unknown. In this project, Dr. Zhang and her research group are using Mtb WhiB1 as the model protein and a suite of high-end structural, spectroscopic, and molecular tools to advance mechanistic understanding of the [4Fe-4S] cluster-dependent control of protein folding and dynamics; the NO-selectivity of the [4Fe-4S] cluster-based transcription factors; and the mode of action of the WhiB1-like monomeric transcription factors.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.