Modern fluorescence imaging technologies such as super-resolution microscopies require novel fluorescent labeling tags possessing nonconventional optical features, including light-controlled turn-on/off of the fluorescence. Our previous reports have demonstrated the ability to engineer hCRBPII to bind a myriad of fluorescent dyes and tune their optical properties. Based on these earlier reports, the goal of this Ph.D. research was to find novel photo-controlled pathways for fluorescence activation of hCRBPII bound fluorophore. In the past two decades, tremendous effort has been invested in the optimization and derivatization of GFP-like fluorescent proteins (FPs). This includes the discovery of photoactivable fluorescent protein (PAFP) variants that becomes fluorescent or change color when they are triggered with light. In contrast to the conventional fluorescent protein, which is permanently fluorescent, photoactive proteins become fluorescent only at the site of interest. In this context, fusion protein which uses synthetic dyes for its optical phenomena provides a broader chemical space for tailoring desired optic features including spectral wavelengths, brightness, stability, and many more photophysical and/or photochemical functionalities. To achieve light-controlled fluorescence activation, two different strategies have been applied here-(1) a cysteine residue containing sulfur was engineered inside hCRBPII, which can participate in a reversible addition with the fluorophore. Utilizing spectroscopic analyses along with X-ray crystallographic studies, we demonstrated that conjugation via Michael addition of cysteine with a coumarin analog that creates a non-fluorescent complex. UV illumination reverses the conjugation, yielding a fluorescent species, presumably through a retro-Michael process. This series of events can be repeated between a bound and non-bound form of the cysteine reversibly, resulting in the ON-OFF control of fluorescence. The details of the mechanism of photoswitching were illuminated by recapitulation of the process in light-irradiated single crystals, confirming the mechanism at atomic resolution. (2) a light induced double proton transfer that results in switching between two spectrally different states of the hCRBPII bound fluorophore. Through spectroscopic and high-resolution structural data, we showed that the protein can be engineered to support selective protonation of the chromophore's aryl amine instead of its imine even at low pH. However, the UV absorbing ammonium ion can be reversibly deprotonated, yielding a highly red-shifted fluorophore upon exposure to UV light. Structural data before and after UV irradiation shows that the light-triggered event alters the protein's interaction with the fluorophore, correlating with the spectral change. The last major endeavor was to develop fluorene based fluorescent dyes with improved optical properties. We have previously reported two fluorene-based dyes, FR0 and FR1V, for fluorescence imaging of the live cells. In this study, effort was made to engineer the dye skeleton to minimize different non-radiating pathways based on literature studies. Spectral data of the new derivatives were collected in different solvents and compared with the previous dyes. We have also been able to demonstrate members of the dyes with red-shifted absorption and emission, high fluorescence QY, and improved water solubility.
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