This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
The use of fluorescence methods to study cellular biology at the molecular level of detail has become a rapidly growing billion dollar industry. Most of these methods depend upon changes in the intensity and/or wavelength of light emitted from a probe molecule when a protein or nucleic acid undergoes a functional process. The PI is performing hybrid quantum mechanical-classical mechanical computations and analysis that lead to a detailed fundamental understanding of the largely unknown underlying molecular physics that cause these changes. Specific focus will be on understanding ultrafast fluorescence intensity decay and wavelength shift experiments on proteins, the spectacular fluctuation of quenching rates seen in single-molecule fluorescence of proteins, and the underlying mechanisms of quenching variation used to monitor protein folding. These are areas of cutting edge experimental work, and the PI works closely with experimental groups to ensure that the computations are relevant and to help with interpreting the experiments. This project builds on previous years of support, which led to unprecedented progress in understanding tryptophan fluorescence wavelength and intensity variability in proteins using electrostatics. The PI's newly acquired ability to realistically evaluate electron transfer coupling elements during dynamics simulations led unexpectedly to an understanding of why wavelength and quenching are often strongly coupled and correlated. The project is now immediately in a position to make insightful contributions to the contested notion that time resolved wavelength shifts speak solely to solvation dynamics, rather than a mixture of solvation dynamics and long term heterogeneity in protein conformation. In summary, a major goal is to quantitatively predict fluorescence quenching and wavelength shift rates for these systems without empirical parameters, including computing relative fluorescence quenching by tryptophan and tyrosine of flavins, chlorophyll, and fluorescent dyes attached to proteins.
The PI's work has shown the importance of the enormous local electric field strength and direction in determining fluorescence behavior in proteins. Continued effort in these areas is encouraged by the emerging view that the catalytic power of enzymes is largely due to a specifically oriented, preorganized electrostatic environment, whose energy may come from reduction in folding energy. A constant theme from the PI's group has been that an ordered electrostatic environment coupled with large fluctuations is precisely what determines whether fluorescence will be strong or weak, and whether its average wavelength will be short or long. This meshes perfectly with the exciting recent observation by Marcus and others that the temporal behavior of fluctuations in electrostatic field is in common with that of other properties of proteins over the time scale of biological importance. Additionally, electron transfer-based fluorescence quenching is considered an obscure subject within the biological community and in need of a rational means for prediction and understanding. The microscopic details of how electron transfer takes place in proteins and other materials are still an area of active debate. There is a high probability that the methods developed by this research will aid in the design of special purpose fluorescent dyes by predicting the extent of quenching by specific quenchers. More broadly, electron transfer in proteins is enormously important to life, given its central role in photosynthesis, oxidative phosphorylation, and cell redox level. It is equally important in the devices that serve most of society. The PI's teaching interests have led to an integrated grasp of quantum mechanics, kinetics, and thermodynamics, all of which play crucial roles in electron transfer processes. He has established a graduate course in the physical chemistry of electron transfer and works with undergraduates, graduate students, and postdoctoral associates in his laboratory; he has productively mentored graduate students in the laboratories of his collaborators.
