Velamins: green‐light‐emitting calcium‐regulated photoproteins isolated from the ctenophore Velamen parallelum

Ca 2+ ‐regulated photoproteins (CaPhs) consist of single‐chain globular proteins to which coelenterazine, a widely distributed marine luminogenic substrate (the luciferin), binds along with molecular oxygen, producing a stable peroxide. Upon Ca 2+ addition, CaPhs undergo conformational changes leading to the cyclization of the peroxide and the formation of a high‐energy intermediate. Subsequently, its decomposition yields coelenteramide in an excited state and results in the emission of a flash of light. To date, most known CaPh systems emit blue light (λ max 465–495 nm), except for two bolinopsin isospecies that emit green light (λ max 500 nm). Here, we report the cloning and functional characterization of wild‐type CaPhs capable of emitting green light: velamins, isolated from the bioluminescent ctenophore Velamen parallelum . Ten unique photoprotein‐like sequences were recovered and grouped in three main clusters. Representative sequences were cloned, expressed, purified, and regenerated into the active His‐tagged α‐, β‐, and γ‐velamins. Upon injection of a calcium‐containing buffer into the velamin, a flash of green light (λ max 500–508 nm) was observed across pH values ranging from 7 to 9. Whilst α‐velamin isoforms exhibited the highest light emission activity, β‐ and γ‐velamins were found to be more thermostable at higher temperatures. Velamins are the wild‐type CaPhs with the longest‐wavelength light emission yet reported, making them an excellent model for investigating spectral modulation mechanisms in photoproteins.