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.

该项目将增加对控制酶活性的新发现的机制的理解，该机制涉及线性自组件的形成（即细丝）。特别是，将发现这种行为的原因以及这种行为有利的情况。这种理解将有助于理解正常生物学中的酶功能，也有助于许多不同的疾病。该行为也可以用于新应用，例如生物技术，材料科学和创建新诊断工具。对社会的更广泛影响包括在高中，本科，研究生和博士后水平的科学方法和方法中培训学生的机会。宣传和招聘工作将是为了扩大培训的机会，以包括来自科学不足社区的成员。该项目的总体科学目标是通过均匀的线性聚合（或细丝）发现调节酶活性的目的和优势。具体而言，将使用SGRAI酶系统测试该机制在快速激活中的假设以及仅在特定反应条件下对二级底物的底物特异性方面的特异性。此外，将测试从结构研究中得出的假设，即丝状如何激活DNA裂解的酶，以及两种类型的底物的DNA序列如何通过控制丝状来控制酶活性。最后，该研究将被扩展，以包括第二丝形成酶，磷酸果糖激酶-1，以开发完整的动力学模型，以便可以鉴定出对这种酶调节的丝状丝的关键优势。要使用的方法包括从单个周转反应得出的酶活性数据的全局动力学建模，并通过更改FRET信号或放射性标记的底物转换为产品而检测到。结构研究将利用最新的冷冻电子显微镜和X射线晶体学方法。诱变将用于创建酶的变体以检验特定的假设，以及用于测量反应性和底物特异性的DNA裂解或果糖磷酸化测定法。该奖项反映了NSF的法定任务，并通过使用该基金会的知识分子优点的支持值得评估。和更广泛的影响审查标准。