Velamins: green‐light‐emitting calcium‐regulated photoproteins isolated from the ctenophore <i>Velamen parallelum</i>

Ca²⁺-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 Ca²⁺ 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.