CAREER: Structure And Function Of Sulfite Reductase Teach About Fundamental Biology

智力上的优点硫磺的减少是生命的核心，因为它提醒人们在氧气能源货币出现之前，地球是一种残留的提醒。硫化物形式的有机硫在所有生物体中仍然是一种基本的营养物质，但高等真核生物无法减少无机硫(IV)，因此依赖细菌、古菌、酵母和植物来生产可用硫。亚硫酸盐的六电子还原为硫化物是生物地硫循环的中心，由单一酶亚硫酸盐还原酶(SIR)催化。这种“大容量”的电子转移是硫和氮循环所独有的，是生化反应的一个强有力的例子，在这种反应中，大自然比人类化学工程师更多才多艺。总之，SIR在营养循环中的中心地位及其独特的生物化学构成了一个令人信服的案例，即对其机制和结构的研究为理解我们的自然环境奠定了重要的基础。该项目的目标是通过实验剖析氧化还原酶SIR在硫磺还原过程中电子转移的化学和生物学，并通过实验室和课堂上的综合教育推广来扩大这项研究的影响。实验结果将通过将来自X射线结晶学和单粒子低温电子显微镜(Cryo-EM)的多分辨率信息与生化分析相结合，促进对硫还原机理的了解。具体地说，有两个领域正在调查中。首先，将通过诱变、SIR活性分析和X射线晶体结构分析来探讨特定氨基酸在SIR活性中的作用。其次，将通过多角度光散射、低温EM成像和单颗粒3DEM分析来探索SIR亚单位组装，以了解驱动硫还原的蛋白质间化学。更广泛的影响应用结构技术回答有关环境细菌学的基本问题，通过提供对硫磺生物地质循环的调节机制的洞察，使整个社会受益。此外，探索硫磺还原路径为研究和教学一体化提供了独特的平台。拟议的外联部分使用基于假设驱动的研究的多方面课堂练习，以吸引一系列学生参与。具体地说，从高中到博士后研究人员的学生将被瞄准加入实验室，在那里他们将接触到结构生物学的研究。佛罗里达州立大学是全球约20台泰坦·克里奥斯低温传输电子显微镜的所在地，对这一独特仪器的培训是拟议研究的组成部分。此外，应用结构技术依赖于来自计算、数学、化学、生物化学和微生物学的各种工具，提供了一系列潜在的研究项目，共同关注回答生物问题。与此同时，佛罗里达州立大学专门为生物学导论一年级课程开发的主动学习模块将加强和加强生物物理学、生物化学、分子生物学和细胞生物学的课堂教学。SIR是一个引人入胜的、有效的课堂练习基础，因为它是一种解释基本生命科学概念的新模式，大多数课程或教科书都没有探讨过。这些与有志于教授K-12科学和技术的学生一起开发的课堂模块进一步扩大了这项提议对改善美国普通科学教育的长期影响。

Intellectual MeritSulfur reduction is central to life as a vestigial reminder of Earth before the emergence of an oxygen-based energy currency. Organic sulfur in the form of sulfide remains an essential nutrient in all organisms, but higher eukaryotes are unable to reduce inorganic sulfur(IV) so rely on bacteria, archaea, yeast, and plants to produce useable sulfur. The six-electron reduction of sulfite to sulfide is central to the bio-geo sulfur cycle and is catalyzed by a single enzyme, sulfite reductase (SiR). This "high-volume" electron transfer is unique to sulfur and nitrogen cycles and represents one powerful example of a biochemical reaction where nature is more versatile than human chemical engineers. Together, the centrality of SiR to nutrient cycles and its unique biochemistry make a compelling case that research into its mechanism and structure lays important groundwork for understanding our natural environment.The goals of this project are to experimentally dissect the chemistry and biology of electron transfer in sulfur reduction by the oxidoreductase SiR and to broaden the impact of this research through integrated educational outreach in the laboratory and classroom. Experimental results will advance knowledge about mechanisms of sulfur reduction by combining information at multiple resolutions from x-ray crystallography and single particle cryogenic electron microscopy (cryo-EM) with biochemical analysis. Specifically, two areas are under investigation. First, the role of specific amino acids in SiR activity will be probed with mutagenesis, SiR activity assays, and x-ray crystallographic structural analysis. Second, the SiR subunit assembly will be explored, with multi-angled light scattering, cryo-EM imaging, and single particle 3DEM analysis, to understand inter-protein chemistry that drives sulfur reduction,. Broader ImpactsApplying structural techniques to answer fundamental questions about environmental bacteriology benefits society at large by providing insight into the mechanisms that regulate the biogeological cycling of sulfur. In addition, exploring sulfur reduction pathways provides a unique platform for integrating research and teaching. The proposed outreach component uses multifaceted classroom exercises based on hypothesis-driven research to engage a range of students. Specifically, students from high school to postdoctoral researchers will be targeted to join the laboratory where they will be exposed to research in structural biology. Florida State University is home to one of approximately 20 Titan Krios cryogenic transmission electron microscopes worldwide and training on this unique instrument is integral to the proposed research. Further, the applied structural techniques rely on diverse tools from computation, mathematics, chemistry, biochemistry, and microbiology, providing a range of potential research projects with a common focus on answering biological questions. At the same time, active-learning modules developed specifically for a first year Introduction to Biology class at Florida State University will reinforce and enhance classroom lessons on biophysics, biochemistry, molecular biology and cellular biology. SiR is an engaging, effective foundation for classroom exercises because it is a novel model to explain basic life science concepts not explored in most lecture courses or textbooks. These classroom modules developed with, and for, students aspiring to teach K-12 science and technology further broadens the long-term impact of this proposal on improving general science education in the United States.