RUI: Site-Directed Mutagenesis Studies of Firefly Luciferase

Branchini MCB 9816898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，这些结构特征能够实现导致发射蓝色至红色可见光的非凡生化过程。生物发光-生物体发光-是一种迷人的自然现象，使儿童着迷，挑战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，这种工具已被应用于有益于人类健康的方式。生物发光的例子可以在自然界的细菌、蘑菇、水母、蚯蚓、蛤、鱼和甲虫中找到，包括大约2,000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，似乎有许多进化起源。通常，荧光素酶、有机底物、分子氧和各种其他辅因子结合联合收割机产生光。本研究的目的是确定特定的活性位点的功能氨基酸残基内的已知的一级序列的荧光素酶蛋白从常见的北美萤火虫萤火虫pyralis。将进行实验以鉴定参与底物结合和/或催化功能的氨基酸，从而导致萤火虫中高效产生光发射。重组DNA技术将用于通过用不同的氨基酸取代目标氨基酸来产生新的酶。选择用于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或接近于Escherichin结合位点，并且可能参与生物发光颜色测定。另一组残基在一大群酶中是不变的，这些酶像荧光素酶一样催化从不同的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个目标。这项研究将有助于更好地了解荧光素酶蛋白的结构与其催化功能之间的关系。此外，这样获得的结果将适用于萤火虫生物发光的基本理解和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-功能特性的阐明对于基本的生物化学过程是重要的，其重要的实际应用超出了生物发光的范围。此外，该项目将在一所文理学院进行，为有效培训本科生提供非常积极的氛围。以前的生物发光工作一直高度吸引学生，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，并将为增加准备充分的毕业生进入研究生课程和专业科学生涯做出积极贡献。

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.