Velamins: the first green-light emitting class of wild-type Ca2+-regulated photoproteins isolated from the ctenophoreVelamen parallelum

Abstract

AbstractCa2+-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, all CaPhs reported produce blue light (λmax465-495 nm). Here, we report the cloning and functional characterization of a novel class of wild-type CaPhs capable of emitting green light: velamins, isolated from the bioluminescent ctenophoreVelamen 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 (λmax500-508 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 only known wild-type Ca2+-regulated photoproteins that exhibit the longest wavelength in light emission, making them a promising model for studying spectral modulation. As a result, velamins hold potential for enhancing the sensitivity of signal detection in analytical systems, particularly when dealing with complex biological matrices.