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Collaborative Research: Structures, Mechanism, and Functional Relevance of Filament Formation by Non-Cytoskeletal Enzymes

合作研究：非细胞骨架酶形成丝的结构、机制和功能相关性

基本信息

批准号：
1934291
负责人：
Nancy Horton
金额：
$ 105.43万
依托单位：
University of Arizona
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1934291&HistoricalAwards=false
关键词：
Collaborative Research Structures Mechanism Functional

项目摘要

This project will increase the understanding of a newly discovered mechanism of controlling enzyme activity, which involves the formation of linear self-assemblies (i.e. filaments). In particular, the reason for this behavior, and those situations where such behavior is advantageous, will be uncovered. Such understanding will aid in the understanding of enzyme function in normal biology, but also in many different diseases as well. The behavior can also be harnessed for new applications such as in biotechnology, material science, and the creation of new diagnostic tools. Broader impacts to society include opportunities for training of students at the high school, undergraduate, graduate, and postdoctoral levels in scientific approaches and methods. Outreach and recruiting efforts will be made to broaden opportunities for training to include members from communities under-represented in science. The overall scientific goal of this project is to discover the purposes and advantages of regulation of enzyme activity via homogeneous, linear polymerization (or filamentation). Specifically, the hypotheses that this mechanism is superior in rapid activation, and in controlling substrate specificity to secondary substrates under only particular reaction conditions, will be tested using the SgrAI enzyme system. In addition, hypotheses derived from structural studies will be tested, namely in how filamentation activates the enzyme for DNA cleavage, and how DNA sequences of the two types of substrates control enzyme activity by controlling filamentation. Finally, the research will be broadened to include a second filament forming enzyme, phosphofructokinase-1, to develop a full kinetic model so that key advantages of filamentation to this enzyme's regulation may be identified. Methodologies to be used include global kinetic modeling of enzyme activity data derived from single turnover reactions and detected via changes in FRET signals or conversion of radiolabeled substrates to products. Structural studies will utilize state-of-the-art cryo-electron microscopy and x-ray crystallographic methods. Mutagenesis will be used to create variants of the enzymes to test specific hypotheses, and DNA cleavage or fructose phosphorylation assays used to measure reactivity and substrate specificity.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.

该项目将增加对新发现的控制酶活性的机制的理解，该机制涉及线性自组装（即细丝）的形成。特别是，将揭示这种行为的原因以及这种行为有利的情况。这样的理解将有助于理解酶在正常生物学中的功能，也有助于理解酶在许多不同疾病中的功能。这种行为也可以用于新的应用，如生物技术，材料科学和新诊断工具的创建。对社会更广泛的影响包括为高中、本科、研究生和博士后学生提供科学方法和方法培训的机会。将开展外联和招聘工作，以扩大培训机会，包括来自科学界代表性不足的社区的成员。该项目的总体科学目标是发现通过均相线性聚合（或聚合）调节酶活性的目的和优点。具体而言，将使用SgrAI酶系统测试该机制在快速活化方面和仅在特定反应条件下控制对次级底物的底物特异性方面具有上级优势的假设。此外，将测试来自结构研究的假设，即双链作用如何激活酶进行DNA切割，以及两种类型底物的DNA序列如何通过控制双链作用来控制酶活性。最后，研究将扩大到包括第二丝形成酶，磷酸果糖激酶-1，开发一个完整的动力学模型，使该酶的调节的关键优势，可以确定。使用的方法包括全球动力学建模的酶活性数据来自单一的营业额反应，并通过FRET信号的变化或放射性标记的底物转化为产品进行检测。结构研究将利用最先进的低温电子显微镜和X射线晶体学方法。诱变作用将被用于创造酶的变体以测试特定的假设，DNA切割或果糖磷酸化测定用于测量反应性和底物特异性。该奖项反映了NSF的法定使命，并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，被认为值得支持。