RUI: Mutagenesis Studies of Old and New Firefly Luciferases

这个项目的目标是增加对生物如何将化学能转化为光的理解。生物发光--生物有机体发出的光--是一种美丽的自然现象，它迷住了儿童，挑战了那些试图理解它的人，并为一种重要的研究工具提供了基础。该项目致力于生产新的荧光素酶蛋白，以改善目前生物发光的研究应用，并推动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有效的研究培训已有记录。现代化的设施和设备为学生和教职员工进行合作研究提供了良好的环境。PI积极指导了37个暑期的本科生研究项目。约有98名本科生与他共事，其中约70%是女性，约10%来自传统上代表性不足的群体，其中约60%的参与者进入了研究生或专业学校。在国际和平研究所实验室工作并对项目做出实质性贡献的学生成为共同作者，并积极参与手稿的撰写和在科学会议上展示他们的结果。其目的是为不同的学生群体提供有意义的研究体验。此外，拟议项目中的几名学生将参与系合作者S-STEM的助学金。所有计划参与者都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生涯做出积极贡献。这个项目的两个主要目标是在之前NSF-Rui赠款的支持下，在相关调查期间制定的。首先是确定一种新克隆的石楠荧光素酶的发射颜色(574 Nm)改变的基础，对低pH红移的抵抗力，以及出人意料的更高的比活性(与最亮的北美萤火虫石楠相比)。主要目的是通过对这两个物种的酶的诱变研究，更好地了解萤火虫排放的颜色和pH稳定性的自然决定因素。PI将全面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。拟议的研究将涉及突变方法，并将通过以下方法研究酶的变体：稳态动力学、生物发光反应产物的定量分析以及包括荧光和生物发光发射技术在内的光谱方法。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)的复合物的晶体结构，并将在结构背景下解释突变数据。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，与目前的技术相比，具有更大的性能便利性和更高的灵敏度。PI将使用两种荧光素酶变体和两种生物发光底物--甲虫荧光素和一种名为苯并噻吩类荧光素的新类似物。此外，PI可以探索使用苯并噻吩类荧光素的含氟衍生物，通过已经设计的合成路线来制造。这两种荧光素酶变体能够产生分离良好的绿色(~520 nm)和红色(~617 nm)发射光谱。PI将使用几种突变策略来产生产生光的酶，并进一步分离发射最大值。分析将在pH、信号稳定剂、裂解剂、底物浓度和光谱过滤器方面进行优化。

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.