The goal of the proposed research project is to provide understanding of the mechanism of photoswitching in fluorescent proteins and to aid in the rational design of new and improved fluorescent proteins. This class of proteins has the ability to be reversibly switched between the 'on'fluorescent bright state and the 'off'dark state via the application of light. These photoswitchable fluorescent proteins have led to revolutionary new imaging techniques that can resolve biological structures on the nanometer scale. Because a low level of illumination is required, these techniques are also much less phototoxic than conventional fluorescence methods, and thus show significant promise for imaging live cells and tissues. For these superresolution microscopy methods to become a reality for imaging living cells, there is a great need for engineering new photoswitchable FPs that emit in a range of colors, are bright, and have tunable switching rates. However, the current understanding of how photoswitching occurs in these proteins is limited. Detailed knowledge of the photoswitching mechanism will undoubtedly aid in the rational design and optimization of new bright photoswitches with a variety of emission colors and switching rates. The proposed work will use quantum dynamic and molecular mechanics simulations of the protein and solvent to model the fluorescent 'on'and 'off'states, and the process of switching between these states. Upon understanding the common mechanistic motif in the known photoswitches, simulations with changes to the protein will be studied to predict possible new photoswitchable fluorescent proteins with shifted absorption/emission maxima, quantum yield, and/or switching rate.
With improved photoswitchable fluorescent proteins, new, noninvasive imaging microscopy techniques will be able to visualize processes in live cells and tissues with unprecedented resolution. This will lead to a deeper understanding of dynamic molecular interactions in biological specimens.
| Isborn, Christine M; Gotz, Andreas W; Clark, Matthew A et al. (2012) Electronic Absorption Spectra from MM and ab initio QM/MM Molecular Dynamics: Environmental Effects on the Absorption Spectrum of Photoactive Yellow Protein. J Chem Theory Comput 8:5092-5106 |
