Roles of Heme and Protein Conformation in Ligand Binding Cooperativity and Selectivity in Bacterial Globin Coupled Sensors

在化学系生命过程化学项目的支持下，宾夕法尼亚州立大学的Emily Weinert和她的研究小组将研究允许细菌对氧气和一氧化氮做出反应的蛋白质的信号机制。细菌对植物、动物、人类和生态系统的健康是必不可少的，但一些物种也会造成有害影响，如感染。为了对环境的变化做出反应，细菌使用蛋白质来感知氧气/一氧化氮，然后改变新陈代谢和表型以实现生存。通过了解细菌对氧气/一氧化氮水平变化的反应，可以开发出改变细菌行为或改造细菌以引发特定功能的方法。拟议的研究将表征传感蛋白如何识别和响应氧气和/或一氧化氮，并将利用这一知识来研究重新连接细菌调控的可能性。该项目将为学生提供蛋白质化学、光谱学和结构质谱学方面的专门培训。此外，推广计划将把细菌传感和信号传递的研究纳入教学计划，利用现成的材料进行实验，并为高中生和本科生制作棋盘游戏。通过在线和在宾夕法尼亚州立大学周围农村社区的高中演讲传播推广材料，预计将使研究计划的结果到达广泛的受众手中。这项研究项目旨在表征细菌血红素蛋白家族中配体结合的协同性和选择性的机制，称为珠蛋白偶联传感器。珠蛋白偶联传感器蛋白在细菌中广泛分布，通常含有参与环状双GMP代谢的输出结构域，控制着生物膜的形成、运动性和毒力等表型。将使用一系列技术，包括平衡配体结合、酶动力学、定点突变、共振拉曼光谱和氢-氚交换质谱学，来询问允许血红素结构域之间通信的相互作用以及连接的c-di-GMP代谢结构域上的配体选择效应。在细菌中的研究将询问配体结合的协同性和选择性对c-di-GMP信号和细菌内表型的影响。这项研究的结果有望为控制合作配体结合、细菌气敏蛋白、c-di-GMP代谢结构域调控以及细菌生物膜形成和毒力调节的机制提供新的信息。这一奖项反映了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。

With the support of the Chemistry of Life Processes program in the Division of Chemistry, Emily Weinert and her research group at Pennsylvania State University will investigate the signaling mechanisms of proteins that allow bacteria to respond to oxygen and nitric oxide. Bacteria are essential for the health of plants, animals, humans, and ecosystems, but some species also can cause detrimental effects, such as infections. To respond to changes in their environment, bacteria use proteins to sense oxygen/nitric oxide and then alter metabolism and phenotypes to enable survival. By understanding how bacteria respond to changing oxygen/nitric oxide levels, methods can be developed to alter bacterial behavior or engineer bacteria to elicit a specific function. The proposed research will characterize how sensing proteins recognize and respond to oxygen and/or nitric oxide and will use this knowledge to investigate the possibility of rewiring bacterial regulation. This project will provide specialized training for students in protein chemistry, spectroscopy, and structural mass spectrometry. In addition, the outreach program will integrate research on bacterial sensing and signaling into lesson plans, experiments using readily available materials, and a board game for high school and undergraduate students. Dissemination of the outreach materials online and through presentations at high schools in rural communities surrounding Penn State University is expected to allow the results of the research program to reach a wide audience.This research project seeks to characterize the mechanisms of ligand binding cooperativity and selectivity within a family of bacterial heme proteins, termed globin coupled sensors. Globin coupled sensor proteins are widely distributed within bacteria and often contain output domains involved in cyclic di-GMP metabolism, which controls phenotypes such as biofilm formation, motility, and virulence. A range of techniques, including equilibrium ligand binding, enzyme kinetics, site-directed mutagenesis, resonance Raman spectroscopy, and hydrogen-deuterium exchange mass spectrometry will be used to interrogate the interactions that allow for communication between heme domains and ligand-selective effects on the linked c-di-GMP metabolic domains. Studies in bacteria will interrogate the effects of ligand binding cooperativity and selectivity on c-di-GMP signaling and phenotypes within bacteria. Results from this study are expected to provide new information about the mechanisms controlling cooperative ligand binding, bacterial gas-sensing proteins, c-di-GMP metabolic domain regulation, and modulation of bacterial biofilm formation and virulence.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.