Structural heterogeneity in biliverdin modulates spectral properties of Sandercyanin fluorescent protein
Sandercyanin, a blue homo-tetrameric lipocalin protein purified from Canadian walleye (Stizostedion vitreus), is the first far-red fluorescent protein reported in vertebrates. Sandercyanin binds non-covalently to biliverdin IXα (BLA) and fluoresces at 675nm on excitation at 375nm and 635nm. Sandercyanin fluorescence can be harnessed for many in vivo applications when engineered into a stable monomeric form. Here, we report the spectral properties and crystal structures of engineered monomeric Sandercyanin-BLA complexes. Compared to wild-type protein, monomeric Sandercyanin (~18kDa) binds BLA with similar affinities and show a broad red- shifted absorbance spectra but possess reduced quantum efficiency. Crystal structures reveal D-ring pyrrole of BLA rotated around the C14-C15 bond, which is stabilized by neighboring aromatic residues and increased water-mediated polar contacts in the BLA-binding pocket. A tetrameric Sandercyanin variant (Tyr-142-Ala) co-displaying red- and far-red absorbing states, and reduced fluorescence shows similar conformational changes in BLA binding pocket. Our results suggest that D-ring flexibility of BLA and its rearrangement reduces the fluorescence quantum-yield of monomeric Sandercyanin. Structures of monomeric Sandercyanin could be utilized as prototypes to generate bright BLA-inducible fluorescent proteins. Further, our study postulates a mechanism for modulating photo-states in BLA- bound lipocalins, known only in phytochromes till date.