Project Summary We began our program by re-engineering members of the Fatty Acid Binding protein family to be mimics of rhodopsin, through rational design principles. In the process of achieving these goals we became convinced that the characteristics of these fantastically stable proteins rendered them ideal vehicles for a variety of other applications. With their small size, large binding pocket that could accommodate a variety of unrelated structures, high expression yield, resistance to structural misfolding due to mutations, and propensity for crystallization, we found these proteins as ideal tools for sensor and imaging applications. The story of their development into unique fluorescent proteins continues with this proposal as a result of two fundamentally important observations, translated to the two major aims of this grant. First, having suitably engineered the binding pocket, and chosen the appropriate chromophore as a partner, we demonstrate the creation of a protein/chromophore complex as an imine, which upon photo-irradiation experiences Excited State Proton Transfer (ESPT) to generate the iminium. Critical to the design of the chromophore is that iminium protonation generates a highly conjugated system capable of Intramolecular Charge Transfer (ICT). ICT fluorophores are typically red-shifted and highly fluorescent. Thus, photo-irradiation of a blue absorbing complex leads to an excited red- shifting species, which fluoresces with a Large Stokes Shift (LSS). Second, we have realized the ability to create a parallel suite of photo-switchable fluorochromes, where the fluorescence output can be rapidly and photo-chemically switched between `ON' and `OFF' states. Such fluorescent systems are the essential tools required for ultra-high resolution microscopy, a technology that has the potential to revolutionize our understanding of biological phenomena if the proper fluorochromes can be developed. They are also essential in biological imaging applications that require spatio-temporal control. The approach to these goals involves the precise, structure-based design and optimization of both protein and fluorophore to find the ideal system. We will optimize ESPT of a protein/chromophore complex as a photobase, a photoacid, and also in what we suggest to be a dual-ESPT mode, requiring both photoacid and photobase activity during the single photo-excitation event. This would convert a ground state neutral imine to a zwitterionic, highly polarized, conjugated excited state that will emit far in the red from the wavelength of excitation. The design of photo- switching fluorophores married to the appropriate protein environment that supports and promotes the structural change in the chromophore, will optimize the characteristics necessary for obtaining a desired photo-switch, such as red-shifted emission and high brightness. Furthermore, `ON' and `OFF' kinetics will be optimized, as rapid rates are advantageous in many imaging applications.