Excited State Proton Transfer in Fluorescent Proteins

荧光蛋白中的激发态质子转移

• 批准号: 1021374
• 负责人: Stephen Remington
• 金额: $ 52.12万
• 依托单位: University of Oregon Eugene
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1021374&HistoricalAwards=false
• 关键词: Excited; State; Proton; Transfer; Fluorescent

Intellectual MeritThis research will characterize and seek to understand the geometric factors that control the rates of proton transfer, one of the most fundamental chemical reactions relevant to biological chemistry. Ultrafast fluorescence emission spectroscopy, ultrafast stimulated Raman spectroscopy and atomic resolution crystallography will be used to investigate excited state proton transfer pathways in green and red fluorescent proteins. Within the chromophore cavities a well-defined proton wire exists, linking the hydroxyl group of the chromophore to a carboxylate acceptor. Proton transfer from the chromophore to the acceptor will be initiated by an actinic flash and the process will be followed on femtosecond to nanosecond time scales by absorbance, ultrafast fluorescence or stimulated Raman spectroscopy. Site directed mutagenesis will be used to substitute groups within the pathway and atomic resolution crystallography will allow determination of the arrangement of the participating atoms. The proton transfer rates, their temperature dependence and atomic motions of the chromophore will be reported and linked to the observed structural features. As a practical spinoff of the research, red fluorescent biosensors will be developed to report on the thiol/disulfide equilibrium within living cells. Due to superior tissue penetration by red light, the new probes will be very useful to biologists for noninvasive studies in animals. Broader ImpactIn the broader view, the results of this research will be important to understand proton transfer processes in many types of vital biological processes, ranging from energy transduction to enzymatic reactions. The data will be useful to validate theoretical efforts to predict the rates of proton transfer processes and the research itself will drive the development of new techniques in ultrafast spectroscopy. The interdisciplinary nature of the effort and the great variety of experimental techniques will provide excellent training for undergraduates, graduate students and postdoctoral researchers. Due to their visual appeal, ease of production and isolation combined with exceptional physical stability, fluorescent proteins are popular as laboratory teaching materials at high school and university levels. Thus, as part of the outreach effort, animated illustrations of research results and proposed molecular motions will be disseminated to the interested public via the Internet, in the form of instructional videos and textual materials.

智力MeritThis研究将表征和寻求理解控制质子转移速率的几何因素，质子转移是与生物化学相关的最基本的化学反应之一。超快荧光发射光谱、超快受激拉曼光谱和原子分辨晶体学将用于研究绿色和红色荧光蛋白中激发态质子转移途径。在发色团空腔内存在明确的质子线，将发色团的羟基连接到羧酸盐受体。从发色团到受体的质子转移将由光化闪光引发，并且该过程将通过吸收、超快荧光或受激拉曼光谱在飞秒到纳秒的时间尺度上进行。定点诱变将用于取代途径内的基团，原子分辨率晶体学将允许确定参与原子的排列。质子转移率，它们的温度依赖性和原子运动的生色团将被报告，并与观察到的结构特征。作为该研究的实际副产品，将开发红色荧光生物传感器，以报告活细胞内的硫醇/二硫化物平衡。由于红光具有优越的上级组织穿透性，新探针将对生物学家进行动物非侵入性研究非常有用。更广泛的影响从更广泛的角度来看，这项研究的结果将是重要的了解质子转移过程中的许多类型的重要生物过程，从能量转导酶促反应。这些数据将有助于验证预测质子转移过程速率的理论努力，研究本身将推动超快光谱学新技术的发展。这项工作的跨学科性质和各种各样的实验技术将为本科生、研究生和博士后研究人员提供良好的培训。由于它们的视觉吸引力，易于生产和分离，加上特殊的物理稳定性，荧光蛋白在高中和大学水平的实验室教学材料中很受欢迎。因此，作为推广工作的一部分，将通过互联网以教学录像和文字材料的形式向感兴趣的公众传播研究结果和拟议分子运动的动画插图。