Biliverdin IXa (BV) binds to several prokaryotic and eukaryotic proteins. How nature exploits the versatility of BV's properties is not fully understood. Unlike free BV, the Sandercyanin fluorescent protein bound to BV (SFP-BV) shows enhanced red fluorescence (675 nm) on excitation in the UV region (380 nm). Site-directed mutagenesis showed that the BV complex of two SFP variants, F55A and E79A, resulted in the loss of red fluorescence. Crystal structures of the complexes of these proteins with BV show the absence of stacking interactions of the F55 phenyl ring with BV. BV changes from ZZZssa conformation in the wild-type to ZZZsss conformation in the variants. In the nonfluorescent mutants, the lowest excited state is destabilized, resulting in nonradiative decay.

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.

Biliverdin IX-alpha undergoes rotation around the D-ring pyrrole and displays a broad far-red absorbance on binding to monomeric Sandercyanin variant (orange) compared to the wild-type tetrameric protein (cyan).