Biosynthesis of Triplet States via Luciferase

在化学系生命过程化学（CLP）项目的支持下，加州大学河滨分校化学系的Michael C . Pirrung、Christopher J . Bardeen和生物工程系的廖佳雨正在研究利用萤火虫的生物发光系统来产生高能态。这种状态在生物系统中具有独特的化学反应性，可以将普通的氧气转化为杀死细胞的氧气。由于该系统依赖于一种可以通过基因工程改造的蛋白质，因此细胞杀伤可以作为对环境的动态反应加以控制。原则上，这个工具可以用于合成生物学和遗传逻辑电路。基础科学将会进步，因为目前还没有可控的生物方法来产生如此高的能量状态。该项目将通过为学生提供跨学科培训和扩大在科学领域代表性不足的群体的参与来造福社会。本项目将专注于利用生物发光来有效地产生三重态激发态，这是生物发色团通常通过光激发实现的过程。这项研究将满足单线态氧（一种参与氧化应激和免疫反应的活性氧）的可控生物生成的需求。研究人员将通过开发成对的荧光素酶突变蛋白和合成的荧光素类似物来实现这一目标，这些荧光素类似物可以产生三重态，并通过致敏作用产生单线态氧，这是一种难以通过生物/遗传控制产生的物种。基于荧光素酶突变体的基因表达，非天然荧光素-荧光素酶对将有助于通过在体外或体内消融特定细胞来修饰生物学。这些目标将涉及非天然荧光素酶底物的产生，其被小萤荧光素酶的接受度通常比荧光素低得多。为了最大限度地利用这些类似物，将采用蛋白质工程技术对酶进行修饰。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemistry of Life Processes (CLP) Program in the Division of Chemistry, Michael C Pirrung, Christopher J Bardeen both of the Department of Chemistry and Jiayu Liao of the Department of Bioengineering at the University of California-Riverside are studying the use of a bioluminescent system of the firefly to generate high energy states. Such states have unique chemical reactivity in biological systems and can convert ordinary oxygen into a species that kills cells. Because the system depends on a protein that can be genetically engineered, cell killing can be controlled as a dynamic response to conditions. This tool could, in principle, be exploited in synthetic biology and genetic logic circuits. Fundamental science will be advanced because no controlled means to generate such high energy states biologically is currently available. This project will benefit society via the provision of interdisciplinary training for students and the broadening of participation by groups underrepresented in science.This project will focus on using bioluminescence to efficiently generate triplet excited states, a process usually achieved for biological chromophores by photoexcitation. This research will fulfill a need for the controlled biological creation of singlet oxygen, a reactive oxygen species (ROS) involved in oxidative stress and the immune response. The investigators will achieve this goal by developing pairs of luciferase mutant proteins and synthetic luciferin analogs that can generate triplet states and, via sensitization, singlet oxygen, a species otherwise difficult to produce with biological / genetic control. Unnatural luciferin-luciferase pairs will be useful to modify biology by ablation of specific cells in vitro or in vivo based on gene expression of luciferase mutants. These goals will involve generation of non-native luciferase substrates, whose acceptance by wt firefly luciferase is typically much lower than luciferin. To maximize utility of these analogues, modification of the enzyme using protein engineering techniques will be undertaken.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.