Photodynamics and Maturation of Coral Fluorescent Proteins

时间分辨晶体学和动力学研究将用于研究amFP595的不寻常的光循环，amFP595是“点燃荧光蛋白”，一种从珊瑚礁生物中分离出来的类似gfp的色蛋白。在不同温度下，利用光泵浦和同步辐射，以非常高的分辨率分析了从黑暗非荧光态到荧光态的可逆光诱导转变及其随后在黑暗中的缓慢弛豫。该研究将确定蛋白质基质中发色团结构转变的性质，中间状态的性质，并确定蛋白质基质如何控制发色团的基态构象和激发态衰变途径。诱变结合Arrhenius图分析将用于研究所选侧链对荧光跃迁构象稳定性、暗弛豫速率和可逆性的能量贡献。这些研究将结合两种非常密切相关的荧光蛋白（蓝色荧光amFP486和黄色荧光zFP538）的有限晶体学分析和诱变，以阐明成熟行为差异的结构基础。据推测，在每种蛋白质中都形成了相同的化学中间体，但由于靠近发色团的少量蛋白质侧链的微妙影响，它们的最终命运不同。虽然这项研究与人类视觉中的光动力过程直接相关，但该项目的一个潜在实际成果是开发一种用于研究活细胞内物质运输的瞬态荧光标签。更广泛的影响：拟议研究的结果预计将产生广泛的影响，因为它们将与许多生物分子的光物理学有关，例如与视觉和光合作用有关的分子。该结果还可以为许多研究人员使用的小分子和蛋白质荧光团观察到的瞬态光/暗切换提供见解。从长远来看，从这项工作中获得的理解可以用来改善荧光蛋白的特性，作为在亚细胞和整个生物体水平上研究生物发育和动态行为的工具。该方法将包括来自物理化学和分子生物学的多种技术，因此将为研究生和博士后研究人员提供优秀的多学科培训。由于其视觉吸引力，易于生产和分离，加上特殊的物理稳定性，荧光蛋白形成了高中和大学水平实验课程的优秀教学资源。

Time resolved crystallographic and kinetic studies will be used to investigate the unusual photocycle of amFP595, the "kindling fluorescent protein", a GFP-like chromoprotein isolated from reef organisms. The reversible light-induced transition from the dark nonfluorescent state to the fluorescent state and its subsequent slow relaxation in the dark will be analyzed at very high resolution using optical pumping and synchrotron radiation in various temperature regimes. The research will determine the nature of the structural transitions of the chromophore within the protein matrix, the nature of intermediate states and determine how the protein matrix controls both the ground state conformation and the excited state decay pathways of the chromophore. Mutagenesis combined with Arrhenius plot analysis will be used to study the energetic contributions of selected side chains to conformational stability, dark relaxation rates and reversibility of the fluorescence transition. These studies will be combined with limited crystallographic analysis and mutagenesis of two very closely related fluorescent proteins, blue fluorescent amFP486 and yellow fluorescent zFP538, in order to elucidate the structural basis for the differences in maturation behavior. It is postulated that the same chemical intermediates are formed in each protein, but suffer different final fates, due to the subtle effects of a small number of protein side chains close to the chromophore. While the research is directly relevant to the photodynamic processes involved in human vision, a potential practical outcome of the project is the development of a transient fluorescent tag for use in studies of material transport within living cells.Broader Impacts: The results of the proposed studies are expected to have broad impact as they will be relevant to the photophysics of many biological molecules, such as those involved in vision and photosynthesis. The results may also provide insight into the transient light/dark switching observed for small molecule and protein fluorophores used by many researchers. In the long term, the understanding gained from this work can be used to improve the properties of fluorescent proteins for use as tools for the study of the development and dynamic behavior of living creatures on both the subcellular and whole organism levels. The methodology will include a great variety of techniques drawn from physical chemistry and molecular biology and will thus provide excellent multidisciplinary training for graduate students and postdoctoral researchers. Due to their visual appeal, ease of production and isolation combined with exceptional physical stability, fluorescent proteins form an outstanding teaching resource for laboratory courses at the high school and university levels.