RUI: Site-Directed Mutagenesis Studies of Firefly Luciferase

BranchiniMCB 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，该酶能够实现导致发射从蓝色到红色的可见光的非凡生化过程。生物发光--生物有机体发出的光--是一种迷人的自然现象，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，这种工具已经以有利于人类健康的方式得到了应用。生物发光的例子在整个自然界的细菌、蘑菇、水母、蚯蚓、文蛤、鱼和甲虫中都可以找到，包括大约2000种萤火虫。从生化角度来看，生物体将化学能转化为可见光的过程是不同的，似乎有许多进化的起源。通常，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石楠荧光素酶蛋白初级序列中特定活性部位氨基酸残基的功能。将进行实验，以确定参与底物结合和/或催化功能的氨基酸，从而在萤火虫中高效地产生光发射。重组DNA技术将被用来通过用不同的氨基酸替换目标氨基酸来创造新的荧光素酶。选择突变的氨基酸包括两个组，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部位或附近，并可能参与生物发光颜色的测定。另一组残基在一大组酶中是不变的，这些酶像荧光素酶一样，催化从一组不同的羧酸底物形成腺苷。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理解荧光素酶蛋白的结构与其催化功能之间的关系。此外，这些结果将适用于对萤火虫生物发光的基本理解，以及生物有机体将化学能转化为光的基本过程。萤火虫荧光素酶的结构和功能特性的阐明对于基本的生化过程是重要的，其重要的实际应用超出了生物发光的领域。此外，该项目将在一所文科学院进行，为有效培养本科生提供非常积极的氛围。以前的生物发光工作对学生具有很高的吸引力，而且应该继续如此。所有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职业生涯的准备充分的毕业生数量做出积极贡献。

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.