RUI: Site-Directed Mutagenesis Studies of Firefly Luciferase

RUI：萤火虫荧光素酶的定点诱变研究

• 批准号: 9816898
• 负责人: Bruce Branchini
• 金额: $ 26万
• 依托单位: Connecticut College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9816898&HistoricalAwards=false
• 关键词: RUI; Site; Directed; Mutagenesis; Studies

BranchiniMCB 9816898The long-term goal of this project is to identify the major structural features of the bioluminescent protein firefly luciferase that enable the extraordinary biochemical process leading to the emission of visible light ranging in color from blue to red. Bioluminescence--the emission of light by living organisms--is a fascinating natural phenomenon that has enchanted children, challenged those who have tried to understand it, and provided the basis for an important research tool which has been applied in ways beneficial to human health. Examples of bioluminescence can be found throughout nature in bacteria, mushrooms, jellyfish, earthworms, clams, fish and beetles, including approximately 2,000 species of fireflies. The processes by which organisms convert chemical energy into visible light are diverse from a biochemical standpoint and appear to have many evolutionary origins. Generally, a luciferase enzyme, an organic luciferin substrate, molecular oxygen and various other cofactors combine to produce light. This study is designed to identify the functions of specific active site amino acid residues within the known primary sequence of the luciferase protein from the common North American firefly Photinus pyralis. Experiments will be carried out to identify amino acids involved in substrate binding and/or catalytic function leading to the highly efficient generation of light emission in the firefly. Recombinant DNA techniques will be used to create new luciferases by substituting different amino acids for the targeted ones. Amino acids selected for mutation comprise two groups, although their functions may be interrelated. The first group of amino acid residues are very likely at or near the luciferin binding site and may be involved in bioluminescence color determination. The other group of residues are invariant among a large group of enzymes which, like luciferase, catalyze the formation of adenylates from a diverse group of carboxylic acid substrates. Preliminary work has enabled the selection of the first targets for mutagenesis studies. This study should contribute to a better understanding of the relationship between the structure of the luciferase protein and its catalytic functions. Furthermore, results so obtained will be applicable to a basic understanding of firefly bioluminescence and the fundamental process by which living organisms convert chemical energy into light. The elucidation of firefly luciferase structure-function properties is important to fundamental biochemical processes with significant practical applications extending beyond the realm of bioluminescence. Moreover, the project will be carried out at a liberal arts college providing a very positive climate for the effective training of undergraduate students. Previous bioluminescence work has been highly appealing to students and should continue to be so. All of the student and professional participants will be involved 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.

该项目的长期目标是确定生物发光蛋白萤火虫荧光素酶的主要结构特征，该特征使非凡的生化过程能够发射从蓝色到红色的可见光。生物发光——生物体发出的光——是一种迷人的自然现象，它使孩子们着迷，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，这种研究工具已被应用于有益于人类健康的方面。生物发光的例子可以在自然界的细菌、蘑菇、水母、蚯蚓、蛤蜊、鱼和甲虫中找到，其中包括大约2000种萤火虫。从生物化学的角度来看，生物体将化学能转化为可见光的过程是多种多样的，似乎有许多进化的起源。一般来说，荧光素酶、有机荧光素底物、分子氧和各种其他辅助因子结合在一起产生光。本研究旨在鉴定北美常见萤火虫Photinus pyralis荧光素酶蛋白已知一级序列中特定活性位点氨基酸残基的功能。实验将进行，以确定氨基酸参与底物结合和/或催化功能，导致在萤火虫高效产生发光。重组DNA技术将通过替换不同的氨基酸来产生新的荧光素酶。选择突变的氨基酸包括两组，尽管它们的功能可能是相互关联的。第一组氨基酸残基很可能位于荧光素结合位点或附近，并可能参与生物发光颜色的测定。另一组残基在一大类酶中是不变的，这些酶如荧光素酶，催化各种羧酸底物形成腺苷酸。初步工作已经能够选择诱变研究的第一批靶点。本研究将有助于更好地理解荧光素酶蛋白的结构与其催化功能之间的关系。此外，获得的结果将适用于对萤火虫生物发光的基本了解以及生物体将化学能转化为光的基本过程。萤火虫荧光素酶的结构功能特性的阐明对基本生化过程具有重要意义，其实际应用已超出生物发光领域。此外，该项目将在一所文理学院进行，为有效培养本科生提供了非常积极的氛围。以往的生物发光工作一直对学生有很大的吸引力，并应继续如此。所有的学生和专业参与者都将参与现代主流生物发光研究，并将为增加进入研究生课程和专业科学事业做好准备的毕业生人数做出积极贡献。