Regulatory and Functional Characterization of Modular Photoprotective Proteins in the Context of Cyanobacterial Ecology and Evolution

光合作用生物在光合作用过程中将光能转化为化学能。然而，吸收的光超过了可用于光合作用的光，可能会导致光诱导的损害。因此，光保护机制已经在光合作用有机体中进化，以处理过度的光吸收或压力条件，包括波动的光。蓝藻所使用的光保护机制直到最近才开始被表征。参与蓝藻光保护的一个关键因素是橙色类胡萝卜素蛋白(OCP)，它与荧光恢复蛋白(FRP)共同作用，将多余的光能转化为热量，保护光合作用反应中心免受光损伤，从而维持光合作用效率和生产力。拟议的工作将有助于更好地了解这些蛋白质的变种范围，这些蛋白质存在于生态上不同的蓝藻中。除了用不同的方法培训学生和博士后科学家外，这项研究的更广泛影响还包括了解蓝藻用来保护自己免受过度光吸收和相关光诱导损害的机制。这些知识将加强努力，提高光捕获的效率，减少生物能源或蓝藻生产菌株的光损害。该项目还包括为来自科学界代表性较低群体的本科生提供有组织的实践研究指导，以及参加一年一度的微生物学日活动，该活动在当地一家科学博物馆举行，目标是中小学生。与植物的光保护机制相比，蓝藻的光保护机制直到最近才开始被表征。其中最普遍的是OCP/FRP(橙色类胡萝卜素蛋白/荧光恢复蛋白)系统，它通过耗散捕光天线(藻胆体或PBS)捕获的多余能量来响应强光。OCP是一种可溶的35 kDa蛋白质，它结合一个单一的类胡萝卜素分子。它是已知的唯一一种以类胡萝卜素为唯一发色团的光活性蛋白质。蓝绿色光的吸收导致OCP从暗稳定的橙色形式OCPO转换为光激活的红色形式OCPR。OCPR通过与PBS结合直接参与光保护。从耗能状态的恢复是由玻璃钢催化的。在结构上，OCP由两个结构域组成，一个作为传感器/调节域，而另一个直接起到能量耗散的作用。我们已经确定了编码Fremyella diplosiphon中不同结构域的一系列平行基因，并假设它们反映了OCP的进化，并为Fremyella在动态环境条件下调节光保护提供了灵活性。弗氏杆菌体内方法和分离蛋白的体外分析将与生物信息学结合使用来检验这些假说。除了用不同的方法培训学生和博士后科学家外，这项研究的更广泛影响包括深入了解光保护机制的灵活性，这可能对蓝藻的生物工程有用。

Photosynthetic organisms convert light energy to chemical energy in the process of photosynthesis. However, absorption of light in excess of that which can be used for photosynthesis can lead to light-induced damage. Thus, photoprotective mechanisms have evolved in photosynthetic organisms to deal with excess light absorption or stressful conditions, including fluctuating light. Photoprotective mechanisms used by cyanobacteria have only recently begun to be characterized. A key factor involved in cyanobacterial photoprotection is the Orange Carotenoid Protein (OCP) that works together with the Fluorescence Recovery Protein (FRP) to convert excess light energy to heat to protect the photosynthetic reaction centers from photodamage, thereby maintaining photosynthetic efficiency and productivity. The proposed work will contribute to a greater understanding of the range of variants of these proteins that are present in ecologically distinct cyanobacteria. In addition to training students and postdoctoral scientists in diverse methods, broader impacts of the research include understanding the mechanisms used by cyanobacteria to protect themselves from excess light absorption and associated light-induced damage. Such knowledge will enhance efforts to increase the efficiency of light capture and reduce photodamage in bioenergy or production strains of cyanobacteria. The project also includes structured mentoring in hands-on research for undergraduates from groups underrepresented in the sciences and participation in an annual Microbiology Day at a local science museum targeting primary and middle school students. In contrast to those of plants, the photoprotective mechanisms of cyanobacteria have only recently begun to be characterized. One of the most prevalent, the OCP/FRP (Orange Carotenoid Protein/Fluorescence Recovery Protein) system responds to high light by dissipating excess energy captured by the light harvesting antenna (phycobilisome or PBS). The OCP is a soluble, 35 kDa protein that binds a single carotenoid molecule. It is the only known photoactive protein that uses a carotenoid as its sole chromophore. The absorption of blue-green light causes the OCP to convert from a dark stable orange form, OCPO, to a light-activated red form, OCPR. OCPR directly participates in photoprotection by binding to the PBS. Recovery from the energy-dissipative state is catalyzed by the FRP. Structurally, the OCP consists of two domains, one serving as a sensor/regulatory domain whereas the other functions directly in energy dissipation. We have identified a family of paralogous genes encoding the separate domains in Fremyella diplosiphon and hypothesize that they reflect the evolution of the OCP and that they provide flexibility in tuning photoprotection in Fremyella under dynamic environmental conditions. A combination of in vivo approaches in Fremyella and in vitro analysis of the isolated proteins will be used in conjunction with bioinformatics to test these hypotheses. In addition to training students and postdoctoral scientists in diverse methods, broader impacts of the research include an in-depth understanding of the flexibility of photoprotective mechanisms that may be useful in bioengineering of cyanobacteria.