Structural heterogeneity in biliverdin modulates spectral properties of Sandercyanin fluorescent protein
Bibliographic record
Abstract
Abstract Sandercyanin, a blue homo-tetrameric lipocalin protein purified from Canadian walleye ( Stizostedion vitreus ), is the first far-red fluorescent protein reported in vertebrates(1–3). Sandercyanin binds non-covalently to biliverdin IXα (BLA) and fluoresces at 675nm on excitation at 375nm and 635nm(1). 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. Significance Statement Sandercyanin is a tetrameric red fluorescent protein from a blue variant of walleye ( Stizostedion vitreum) that binds to biliverdin IXα (BLA). Its biophysical properties and structures have been published earlier(1). A bright and stable monomeric Sandercyanin could be utilized as a fusion protein for fluorescence-based applications. Here we report the first structures and spectral properties of fluorescent monomeric Sandercyanin-BLA complexes and describe the molecular basis of modulated spectral properties due to rotated D-ring pyrrole around C14-C15 bond and re-shuffling of BLA-binding pocket. BLA-bound monomeric Sandercyanin could be engineered into brighter variants for in-vivo applications. Our study also reveals an unfamiliar mechanism in BLA-binding lipocalins that regulates red- and far-red absorbance states.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".