Background: Near-infrared (NIR) fluorescent bacteriophytochromes are valuable for optical imaging in mammals. Results: Reversal of one position in the fluorescent phytochrome variant IFP1.4 led to the brightest monomeric NIR phytofluor known. Conclusion: Crystallography shows that limiting motion and changing polarity in the chromophore binding pocket increase fluorescence. Significance: Understanding the source of increased fluorescence in NIR fluorescent phytofluors is essential for further improving these novel imaging tools.

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 IXα 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.

Use of fluorescent proteins to study in vivo processes in mammalian systems begs development of near-infrared (NIR) biomarkers due to superior penetration of the excitation light at longer wavelengths. Bacteriophytochromes (BphPs) that use biliverdin as their chromophore have been engineered to form monomeric NIR biomarkers. Absorption of a red light photon (l=700 nm) leads to isomerization of the C15=C16 bond of BV in the Pr ground state, leading to the second ground state, the far red-light absorbing Pfr. The archetypal NIR phytofluor carried a D207H substitution. The 207 position is important because the main chain carbonyl forms a hydrogen bond with the BV A ring, thus potentially functioning as a proton sink during the photocycle. This variant spurred development of NIR biomarker BPhPs IFP1.4, Wi-Phy, and iRFP. We have solved the structure of IFP1.4 and observe a tightly packed hydrophobic core in the BV binding pocket in contrast to the open pocket in native BphP. Side chains of V173, M174, and V288 form this stabilizing interaction, which is absent in the wild type counterpart because residue 288 is an alanine. We have further disproven the hypothesis that H207 is required for fluorescence, by engineering IFP1.4 D207 and demonstrating it is brighter than any NIR phytofluor described to date. Our research has helped delineate the origin of fluorescence in BphPs and will help phytofluors gain wide spread use as their brightness and resistance to photobleaching are augmented.