RUI: Mutagenesis Studies of Old and New Firefly Luciferases

RUI：新旧萤火虫荧光素酶的诱变研究

• 批准号: 1410390
• 负责人: Bruce Branchini
• 金额: $ 59.94万
• 依托单位: Connecticut College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-15 至 2019-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1410390&HistoricalAwards=false
• 关键词: RUI; Mutagenesis; Studies; Old; New

The goal of this project is to increase the understanding of how living organisms convert chemical energy into light. Bioluminescence--the emission of light by living organisms--is a beautiful natural phenomenon that has enchanted children, challenged those who have tried to understand it, and provided the basis for an important research tool. The project addresses the need to produce novel luciferase proteins to improve current research applications of bioluminescence and to advance the development of new ones. The proposed research will be carried out at a liberal arts college with an established record of effective research training of undergraduate students. Modern facilities and equipment provide an excellent environment for students and faculty to engage in collaborative research. The PI has actively directed undergraduate research projects for 37 summers. Approximately 98 undergraduate students, approximately 70% of whom are women and approximately 10% who are from traditionally underrepresented groups, have worked with him and approximately 60% of these participants entered graduate or professional school. Students who work in the PI's lab and make substantive contributions to a project become co-authors and are actively involved in the writing of manuscripts and presenting their results at scientific meetings. The aim is to offer a meaningful research experience to a diverse group of students. Moreover, several of the students in the proposed program will be participants in a department collaborator's NSF S-STEM grant. All program participants will engage in modern mainstream bioluminescence research and will contribute positively to increasing the numbers of well-prepared graduates for entry into graduate programs and professional scientific careers. Two major objectives are addressed in this project that were developed during related investigations with prior NSF-RUI grant support. The first is to determine the basis for the altered emission color (574 nm), resistance to low pH red shifting, and unexpectedly greater specific activity (compared to the brightest North American firefly Photinus pyralis) of a newly cloned luciferase from Photinus scintillans. The main aim is to better understand the natural determinants of firefly emission color and pH stability through mutagenesis studies of the enzymes from both species. The PI will fully characterize the physical and spectral properties of the new enzyme and undertake biochemical studies to identify key amino acid residues that determine the enhanced properties. The proposed investigation will involve a mutagenesis approach and variants of the enzymes will be studied by: steady state kinetics, quantitative analysis of bioluminescence reaction products, and spectral methods including fluorescence and bioluminescence emission techniques. With collaborator Professor Andrew Gulick, the crystal structure of a complex of the novel luciferase with DLSA, an inhibitor that binds to the active site, will be determined and the mutagenesis data will be interpreted in a structural context. The second objective is to develop new substrates and red-emitting luciferase variants for improved dual color (analyte) reporter gene applications. The plan is to seek new assay methodology with greater convenience of performance and enhanced sensitivity compared to current technology. The PI will use 2 luciferase variants and 2 bioluminescence-producing substrates-- beetle luciferin and a novel analog called benzothiophene luciferin. Additionally, the PI may explore the use of fluorine-containing derivatives of benzothiophene luciferin to be made by synthetic routes that have been designed. The 2 luciferase variants are engineered to produce well-separated green (~520 nm) and red (~617 nm) emission spectra. The PI will use several mutagenesis strategies to produce enzymes that produce light with further separated emission maxima. The assays will be optimized with respect to pH, signal stabilizers, lysing agents, substrate concentrations and spectral filters.

该项目的目标是增加对生物体如何将化学能转化为光的理解。 生物发光--生物体发光--是一种美丽的自然现象，它让孩子们着迷，对那些试图理解它的人提出了挑战，并为一种重要的研究工具提供了基础。 该项目解决了生产新型荧光素酶蛋白的需求，以改善目前生物发光的研究应用，并促进新的荧光素酶蛋白的开发。拟议的研究将在一所文科学院进行，该学院对本科生进行了有效的研究培训。现代化的设施和设备为学生和教师提供了一个良好的环境，进行合作研究。 PI积极指导本科生研究项目37个夏天。大约98名本科生，其中约70%是女性，约10%来自传统上代表性不足的群体，与他一起工作，其中约60%进入研究生或专业学校。 在PI实验室工作并对项目做出实质性贡献的学生成为合著者，并积极参与手稿的撰写并在科学会议上展示他们的成果。 其目的是为不同群体的学生提供有意义的研究经验。此外，拟议计划中的几名学生将参加部门合作者的NSF S-STEM资助。 所有计划参与者将从事现代主流生物发光研究，并将为增加准备充分的毕业生进入研究生课程和专业科学职业的数量做出积极贡献。本项目的两个主要目标是在先前的NSF-RUI资助的相关调查期间开发的。首先是确定的基础上改变发射颜色（574 nm），耐低pH值红移，和意想不到的更大的比活性（相比最亮的北美萤火虫Photinus pyralis）的新克隆的荧光素酶Photinus pyrans。 主要目的是更好地了解萤火虫发射颜色和pH稳定性的自然决定因素，通过诱变研究的酶从两个物种。 PI将充分表征新酶的物理和光谱特性，并进行生化研究，以确定决定增强特性的关键氨基酸残基。拟议的调查将涉及诱变方法和酶的变体将研究：稳态动力学，生物发光反应产物的定量分析，光谱方法，包括荧光和生物发光发射技术。与合作者Andrew Gulick教授一起，将确定新型荧光素酶与DLSA（一种与活性位点结合的抑制剂）复合物的晶体结构，并将在结构背景下解释诱变数据。 第二个目标是开发新的底物和红色荧光素酶变体，用于改进双色（分析物）报告基因的应用。该计划是寻求新的分析方法，与现有技术相比，具有更大的性能便利性和更高的灵敏度。 PI将使用2种荧光素酶变体和2种生物发光产生底物--甲虫荧光素和一种名为苯并噻吩荧光素的新型类似物。此外，PI可能会探索使用苯并噻吩的含氟衍生物通过已设计的合成路线制备。对2种荧光素酶变体进行工程改造，以产生分离良好的绿色（~520 nm）和红色（~617 nm）发射光谱。 PI将使用几种诱变策略来产生产生具有进一步分离的发射最大值的光的酶。将在pH值、信号稳定剂、裂解剂、底物浓度和光谱过滤器方面优化试验。