Excited State Proton Transfer in Fluorescent Proteins

University Of Oregon Eugene, Eugene OR

Investigators

Abstract

Intellectual Merit This 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 Impact In 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.