Abstract
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.
Highlights
Fluorophores active in the near infrared (NIR) attract ongoing attention due to their diverse applications in biomedical research, materials science and related fields
There were no significant changes to the overall structure of WiPhy2 compared to DrCBDmon (RMSD 0.82 Å over all 296 shared Cα atoms including mobile loop regions; Figure 2B)
The BV chromophore is well-ordered with no evidence of a break in electron density for the cysteine connection to the A-ring (Figure 2A)
Summary
Fluorophores active in the near infrared (NIR) attract ongoing attention due to their diverse applications in biomedical research, materials science and related fields. They allow imaging with minimal autofluorescence and light scattering in animals, and deep tissue penetration (Weissleder, 2001). The development of simple, stable, non-toxic, modular, and small molecular weight NIR platforms is of great interest to the biomedical community and has proceeded both in the realm of chemical biology and fluorescent proteins. The promise of genetically encoded NIR fluorescent proteins has, on the other hand, led to a renaissance in research of engineered fluorescent proteins, based both on Green Fluorescent Protein-like β-barrel folds and more recently on bacteriophytochromes (Zhang et al, 2002; Gibbs, 2012; Guo et al, 2014; Marx, 2014)
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