"Our primary goal is to develop fluorescent proteins that span the entire visible spectra, to be used when conventional fluorescent proteins are inadequate. In our system, fluorescence is activated upon coupling of the protein and ligand, such that temporal control can be achieved, whereas intrinsically fluorescent proteins are constitutively on. Additionally, our system does not require oxygen and can therefore find potential uses in obligate anaerobes. Our lab has demonstrated the ability to effectively control the absorption profile of conjugated polyenes. The initial aim of this project was to regulate the emissive properties of bound fluorophores with the same degree of control. This was achieved by the coupling of the solvatochromic fluorophore ThioFluor to hCRBPII mutants. ThioFluor yielded mutants with absorption maxima varying from 501 nm to 705 nm and emission maxima from 613 nm to 744 nm. This is equivalent to regulation over 204 nm in absorption and 131 nm in emission, covering both the red and far-red fluorescence wavelength regimes. Furthermore, we have shown its utility in live-cell imaging in whole cells, and with targeting to the nucleus and extranuclear space; fortuitously, negligible background fluorescence is apparent. In the course of optimizing binding of ThioFluor to hCRBPII, we discovered that it was observed that not only is the protonated Schiff base (PSB) fluorescent, but the Schiff base (SB) is as well. However, while PSB emission wavelength could be altered over 204 nm, SB emission remains nearly constant at 500 nm. Interestingly, select mutants displayed a far-red emission upon SB irradiation, similar to that obtained upon irradiation of the PSB, presumably through protonation in the excited state. This serendipitous discovery leads to more than a 200 nm Stokes shift with high quantum yield (> 60%). One such hCRBPII/ThioFluor complex displays ideal spectroscopic properties including fast iminium formation with a half-life of 1.7 min, low pKa of 5.3 (rendering almost complete SB formation at physiological pH), high quantum yield (0.51) and large Stokes shift (208 nm). This fluorescent protein was successfully used to visualize whole cell fluorescence, as well as targeting to the nucleus. The last major endeavor was to develop a protein-based pH sensor, with the ability to report pH values with high accuracy. We have previously reported an absorptive system that was capable of ratiometric sensing of pH. However, we have now developed a single protein fluorescent ratiometric pH sensor, based on the titration of an acidic residue near the iminium. Standard curves were generated based on the ratio of emissions at two excitation wavelengths, allowing for concentration independent sensing of pH. We have been able to demonstrate its applicability as an in vivo fluorescent pH probe, obtaining a pH value of 6.7 when hCRBPII is targeted to the nucleus of HeLa cells."--Pages ii-iii.
Read
