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

由于铁（Fe）和硫（S）离子都具有高度反应性，因此含有多铁和硫组装体（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方面的作用。Fe-S簇蛋白的适当功能是细菌适应性所必需的。 这些蛋白质通常赋予持久性和抗生素抗性的细菌是致病的，他们的主机。尽管这些Fe-S簇蛋白很重要，但由于使用这些氧敏感蛋白的技术挑战，关于它们在细菌中如何发挥作用及其潜在作用机制的知识存在重大空白。生命过程化学和刺激竞争研究的既定计划（EPSCoR）计划共同资助内布拉斯加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计划，其目标是破译一种非常重要但尚未充分研究的放线菌中Fe-S簇蛋白WhiB 1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，持久的病原体结核分枝杆菌。 这项研究还可以提高利用放线菌生产生物活性分子的能力，从而对农业产生好处。这项研究被整合到一系列旨在招募和培训下一代STEM研究人员的教育和推广活动中，重点关注在科学领域代表性不足的群体。 它还通过与包括K-12，本科生和研究生以及公众在内的受众接触来促进科学素养。WhiB 1是一种基于[4Fe-4S]簇的一氧化氮（NO）传感器，广泛分布于放线菌中，是结核分枝杆菌（Mtb）的必需传感器。WhiB 1与Mtb的NO胁迫反应和休眠启动有关。尽管对这种蛋白质进行了数十年的研究，但在定义WhiB 1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响应细胞NO水平选择性地感测和调节基因表达方面发挥着重要作用。除了关于NO传感和应激反应的细节之外，WhiB 1代表了一个未充分研究的参与信号传导的单体转录因子的大家族，其潜在的作用机制尚不清楚。在这个项目中，Zhang博士和她的研究小组正在使用Mtb WhiB 1作为模型蛋白和一套高端结构，光谱和分子工具，以推进对蛋白质折叠和动力学的[4Fe-4S]簇依赖性控制机制的理解;基于[4Fe-4S]簇的转录因子的NO选择性;该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

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.