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.
One of the most fundamental chemical reactions in biology is proton transfer, that is, the transfer of a naked hydrogen atom from one compound to another. Our long term research interests include study of enzymes that catalyze proton transfers, study of sensory proteins that detect acidic conditions and methods of monitoring pH within cells. Some years ago it was discovered that proton transfer is central to fluorescence emission in certain fluorescent proteins, including the most popular example, Green Fluorescent Protein (GFP). We had previously reengineered GFP to produce noninvasive biosensors for use as tools in cell biology to study pH changes and thiol/disulfide redox reactions within compartments of living cells. In this particular study we conducted both basic research and practical product development. We first made specific mutations in GFP to alter the rates of proton transfer, characterized the resulting molecular structures in atomic detail and in collaboration with physical chemists, used ultrafast spectroscopy to measure the reaction rates and molecular dynamics calculations to study the mechanistic details of proton transfer. Our results showed that GFP is optimized for ultrafast proton transfer, so it was not surprising that mutations slowed transfer rates by factors of up to 350 fold. Several publications advance the field by describing the experimental results and their theoretical interpretation. However, a practical goal of the research was to use the insight gained to create a red fluorescent redox sensor, following the same principles that we used earlier to create redox-sensitive GFPs (roGFPs). The newly created redox sensor, roRFP1, has been functionally tested in vitro and in yeast cells and in principle, allows cell biologists to use two-color imaging to visualize chemical processes in different compartments of the same cell, simultaneously. See Figure 1 for some structural details of roRFP1. An unexpected opportunity arose to analyze the crystal structure of a bacterial acid-sensing protein, tlpB, in collaboration with the Guillemin laboratory at the University of Oregon. tlpB is a key sensory protein in Helicobacter pylori, allowing the bacterium to navigate away from acidic environments in the process of infecting the host. We determined the crystal structure of tlpB at atomic resolution, together with several of its mutants and used these data to propose a detailed mechanism for the acid sensing activity. We further assisted the Guillemin laboratory in studies to demonstrate that this protein is a key sensory element, allowing bacteria to detect and move away from acidic regions in their environment. A schematic illustration of the structure of tlpB, the acid sensor, and a key sensory element (a molecule of urea) is presented in Figure 2. These two studies are expected to have major impacts on their respective fields. In addition to the advancement in understanding basic proton transfer rates, the new redox sensitive, red fluorescent biosensor roRFP1 will be a popular research tool among cell biologists. The elucidation the tlpB structure in atomic detail helps explain for the first time the mechanism of acid sensing and in addition, revealed structural domains that are likely to become the new paradigm for sensory protein structure. The research also provided extensive training in advanced laboratory techniques and methodologies to a number of undergraduate students, graduate students and postdoctoral research associates.
