Excited State Proton Transfer in Fluorescent Proteins

本研究将描述并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率晶体学将用于研究绿色和红色荧光蛋白的激发态质子转移途径。在发色团腔内存在一条明确的质子线，将发色团的羟基与羧酸受体连接起来。质子从发色团到受体的转移将由光化闪光开始，这一过程将通过吸光度、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。位点定向诱变将用于替代途径中的基团，原子分辨率晶体学将允许确定参与原子的排列。质子转移速率、它们的温度依赖性和发色团的原子运动将被报道并与观察到的结构特征联系起来。作为该研究的实际副产品，将开发红色荧光生物传感器来报告活细胞内的硫醇/二硫平衡。由于红光对组织的穿透能力强，这种新型探针对生物学家在动物身上进行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，这项研究的结果对于理解从能量转导到酶促反应等许多重要生物过程中的质子转移过程非常重要。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，荧光蛋白作为高中和大学水平的实验室教材很受欢迎。因此，作为外联工作的一部分，研究结果的动画插图和拟议的分子运动将通过互联网以教学录像和文本材料的形式传播给感兴趣的公众。

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.