Removal of Chromophore-Proximal Polar Atoms Decreases Water Content and Increases Fluorescence in a Near Infrared Phytofluor

Genetically encoded fluorescent markers have revolutionized cell and molecular biology due to their biological compatibility, controllable spatiotemporal expression, and photostability. To achieve in vivo imaging in whole animals, longer excitation wavelength probes are needed due to the superior ability of near infrared light to penetrate tissues unimpeded by absorbance from biomolecules or autofluorescence of water. Derived from near infrared-absorbing bacteriophytochromes, phytofluors are engineered to fluoresce in this region of the electromagnetic spectrum, although high quantum yield remains an elusive goal. An invariant aspartate residue is of utmost importance for photoconversion in native phytochromes, presumably due to the proximity of its backbone carbonyl to the pyrrole ring nitrogens of the biliverdin (BV) chromophore as well as the size and charge of the side chain. We hypothesized that the polar interaction network formed by the charged side chain may contribute to the decay of the excited state via proton transfer. Thus, we chose to further probe the role of this amino acid by removing all possibility for polar interactions with its carboxylate side chain by incorporating leucine instead. The resultant fluorescent protein, WiPhy2, maintains BV binding, monomeric status, and long maximum excitation wavelength while minimizing undesirable protoporphyrin IX alpha binding in cells. A crystal structure and time-resolved fluorescence spectroscopy reveal that water near the BV chromophore is excluded and thus validate our hypothesis that removal of polar interactions leads to enhanced fluorescence by increasing the lifetime of the excited state. This new phytofluor maintains its fluorescent properties over a broad pH range and does not suffer from photobleaching. WiPhy2 achieves the best compromise to date between high fluorescence quantum yield and long illumination wavelength in this class of fluorescent proteins.