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Here, we describe (1) the AlphaFold-based structural modeling approach to identify amino acids of the photoprotein berovin that are crucial for coelenterazine binding, and (2) the production and characterization of berovin mutants with substitutions of the identified residues regarding their effects on the ability to form an active photoprotein under physiological conditions and stability to light irradiation. The combination of mutations K90M, N107S, and W103F is demonstrated to cause a shift of optimal conditions for the conversion of apo-berovin into active photoprotein towards near-neutral pH and low ionic strength, and to reduce the sensitivity of active berovin to light. According to the berovin spatial structure model, these residues are found in close proximity to the 6-(p-hydroxy)-phenyl group of the coelenterazine peroxyanion. [ABSTRACT FROM AUTHOR]

Moreover, this combination provided light signals several orders of magnitude higher than that of the commercial AkaLumine with firefly luciferase, a slower bioluminescence decay time, and 3-fold higher activity of bioluminescence in vivo in bacterial cells as compared to firefly luciferin, owing to the better penetrability of the 6'-(1-pyrrolidinyl) luciferin analogue across bacterial cell membranes. The first genes encoding two-subunit luciferase from luminous bacteria I Vibrio harveyi i , firefly luciferase from I Photinus pyralis i , and Ca SP 2+ sp -regulated photoprotein aequorin from jellyfish I Aequorea victoria i were cloned almost simultaneously, probably due to the availability of these bioluminescent proteins and the highly motivated research teams involved [[1]]. Noteworthy is that, although the properties of copepod luciferases such as excellent thermal stability and bright luminescence are very suitable to use as labels in various in vitro bioluminescence assays, the examples of their applications are few, which is mainly caused by difficulties in producing the correctly folded recombinant copepod luciferases in inexpensive expression systems [[8]]. An interesting feature of the cloning of copepod luciferases is that the first cDNA genes encoding these proteins were isolated applying the functional screening of the relevant cDNA libraries in I Escherichia coli i by colony luminescence with a bioluminescence substrate. [Extracted from the article]

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid [...]

Results indicate that the recombinant obelin from Obelia geniculata shares several characteristics of that of O. longissima protein. Data point out that obelin from these two marine species function as intracellular calcium indicators, considering their sensitivity to magnesium ion, which plays a role in the bioluminescence.

Coelenterazine‐v (CTZ‐v), a synthetic derivative with an additional benzyl ring, yields a bright bioluminescence of Renilla luciferase and its "yellow" mutant with a significant shift in the emission spectrum toward longer wavelengths, which makes it the substrate of choice for deep tissue imaging. Although Ca2+‐regulated photoproteins activated with CTZ‐v also display red‐shifted light emission, in contrast to Renilla luciferase their bioluminescence activities are very low, which makes photoproteins activated by CTZ‐v unusable for calcium imaging. Here, we report the crystal structure of Ca2+‐regulated photoprotein obelin with 2‐hydroperoxycoelenterazine‐v (obelin‐v) at 1.80 Å resolution. The structures of obelin‐v and obelin bound with native CTZ revealed almost no difference; only the minor rearrangement in hydrogen‐bond pattern and slightly increased distances between key active site residues and some atoms of 2‐hydroperoxycoelenterazine‐v were found. The fluorescence quantum yield (ΦFL) of obelin bound with coelenteramide‐v (0.24) turned out to be even higher than that of obelin with native coelenteramide (0.19). Since both obelins are in effect the enzyme‐substrate complexes containing the 2‐hydroperoxy adduct of CTZ‐v or CTZ, we reasonably assume the chemical reaction mechanisms and the yields of the reaction products (ΦR) to be similar for both obelins. Based on these findings we suggest that low bioluminescence activity of obelin‐v is caused by the low efficiency of generating an electronic excited state (ΦS). In turn, the low ΦS value as compared to that of native CTZ might be the result of small changes in the substrate microenvironment in the obelin‐v active site. PDB Code(s): 7O3U; 1QV0, 1QV1, 1EJ3, 4NQG, 3KPX, 1UHK, 1UHJ, 1UHI, 1UHH, 5ZAB, 1EL4, 4MRX. [ABSTRACT FROM AUTHOR]

Nowadays the recombinant Ca2+‐regulated photoproteins originating from marine luminous organisms are widely applied to monitor calcium transients in living cells due to their ability to emit light on Ca2+ binding. Here we report the specific activities of the recombinant Ca2+‐regulated photoproteins—aequorin from Aequorea victoria, obelins from Obelia longissima and Obelia geniculata, clytin from Clytia gregaria and mitrocomin from Mitrocoma cellularia. We demonstrate that along with bioluminescence spectra, kinetics of light signals and sensitivities to calcium, these photoproteins also differ in specific activities and consequently in quantum yields of bioluminescent reactions. The highest specific activities were found for obelins and mitrocomin, whereas those of aequorin and clytin were shown to be lower. To determine the factors influencing the variations in specific activities the fluorescence quantum yields for Ca2+‐discharged photoproteins were measured and found to be quite different varying in the range of 0.16–0.36. We propose that distinctions in specific activities may result from different efficiencies of singlet excited state generation and different fluorescence quantum yields of coelenteramide bound within substrate‐binding cavity. This in turn may be conditioned by variations in the amino acid environment of the substrate‐binding cavities and hydrogen bond distances between key residues and atoms of 2‐hydroperoxycoelenterazine. [ABSTRACT FROM AUTHOR]