Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy.

Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea. Intrinsic steady-state and time-resolved fluorescence spectra and circular dichroism spectra were used to determine the stages of the protein unfolding. Molecular dynamics methods were applied to find the differences in the surroundings of tryptophans in both luciferases. We found that the unfolding pathway is the same for the studied luciferases. However, the results obtained indicate more stable tertiary and secondary structures of P. leiognathi luciferase as compared to enzyme from V. harveyi during the last stage of denaturation, including the unfolding of individual subunits. The distinctions in fluorescence of the two proteins are associated with differences in the structure of the C-terminal domain of α-subunits, which causes different quenching of tryptophan emissions. The time-resolved fluorescence technique proved to be a more effective method for studying protein unfolding than steady-state methods.

Coupling of NAD(P)H:FMN-oxidoreductase and luciferase from luminous bacteria in a viscous medium: Finding the weakest link in the chain.

The study aims at revealing the mechanisms of the viscous medium effects on the kinetic features of NAD(P)H:FMN-oxidoreductase from luminous bacteria (Red), which are exhibited in a single enzyme assay and in coupling with bacterial luciferase (BLuc). Different concentrations of glycerol and sucrose were used to vary the medium viscosity. The activity of Red, alone and in the presence of BLuc, was analyzed, as well as BLuc activity in the presence of Red, whereas in the absence of BLuc, the Red activity was suppressed in viscous medium, and in the presence of BLuc, the increase in Red activity was observed at low glycerol concentrations (5-20 wt%). The interaction of glycerol and sucrose with Red substrates FMN and NADH was studied using absorption spectroscopy and molecular dynamics. Glycerol was found to form hydrogen bonds with the phosphate groups of the substrates, unlike sucrose. A mechanism for the activation of Red in the presence of BLuc in glycerol solutions through the acceleration of FMN reoxidation was proposed. Thus, it was concluded that, under the conditions used, the weakest link of the coupled enzyme system BLuc-Red in viscous medium is the FMN concentration, which depends on Red activity and the medium viscosity.

The Role of Cosolvent-Water Interactions in Effects of the Media on Functionality of Enzymes: A Case Study of Photobacterium leiognathi Luciferase.

A complex heterogeneous intracellular environment seems to affect enzymatic catalysis by changing the mobility of biomolecules, their stability, and their conformational states, as well as by facilitating or hindering continuously occurring interactions. The evaluation and description of the influence of the cytoplasmic matrix components on enzymatic activity are problems that remain unsolved. In this work, we aimed to determine the mechanisms of action of two-component media with cosolvents of various molecular sizes on the complex multi-stage bioluminescent reaction catalyzed by bacterial luciferase. Kinetic and structural effects of ethylene glycol, glycerol, sorbitol, glucose, sucrose, dextran, and polyethylene glycol on bacterial luciferase were studied using stopped-flow and fluorescence spectroscopy techniques and molecular dynamics simulations. We have found that diffusion limitations in the presence of cosolvents promote the stabilization of flavin substrate and peroxyflavin intermediate of the reaction, but do not provide any advantages in bioluminescence quantum yield, because substrate binding is slowed down as well. The catalytic constant of bacterial luciferase has been found to be viscosity-independent and correlated with parameters of water-cosolvent interactions (Norrish constant, van der Waals interaction energies). Crowding agents, in contrast to low-molecular-weight cosolvents, had little effect on peroxyflavin intermediate decay and enzyme catalytic constant. We attributed specific kinetic effects to the preferential interaction of the cosolvents with enzyme surface and their penetration into the active site.

Specific Activities of Hydromedusan Ca2+ -Regulated Photoproteins.

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.

Crystal structure of semi-synthetic obelin-v after calcium induced bioluminescence implies coelenteramine as the main reaction product.

Coelenterazine-v (CTZ-v), a synthetic vinylene-bridged π-extended derivative, is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths. However, Ca2+-regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence. Here, we report the crystal structure of semi-synthetic Ca2+-discharged obelin-v bound with the reaction product determined at 2.1 Å resolution. Comparison of the crystal structure of Ca2+-discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure. However, the drastic changes in CTZ-binding cavity are observed owing to the completely different reaction product, coelenteramine-v (CTM-v). Since CTM-v is certainly the main product of obelin-v bioluminescence and is considered to be a product of the "dark" pathway of dioxetanone intermediate decomposition, it explains the low bioluminescence activity of obelin and apparently of other photoproteins with CTZ-v.

Crystal structure of semisynthetic obelin-v.

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.