Crystal structure of semisynthetic obelin‐v

Abstract

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 Ca 2+ -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 Ca 2+ -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 (Φ FL ) 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 (Φ R ) 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 (Φ S ). In turn, the low Φ S 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.

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

“…5 a). Under excitation at 407 nm we observed redistribution of intensity between the two peaks relative to fluorescence under excitation at 373 nm 15 , which suggest the presence of multiple fluorophores with different absorption spectra (Fig. S1 ).…”

“…In both obelin and obelin- v containing the corresponding 2-hydroperoxyCTZs, the C -terminus effectively caps the substrate-binding cavity, ensuring a solvent-inaccessible and nonpolar environment of the CTZ derivative 15 , 19 . The inaccessibility of the internal substrate-binding cavity of photoproteins to solvent is additionally guarded by the hydrogen bonds formed by Arg21 located in helix A and C -terminal Pro as well as Asp187 and Phe178 25 , 26 .…”

“…Using mutants of Renilla luciferase with bioluminescence maxima at 547 nm in combination with CTZ- v as a luciferin, it was possible to shift bioluminescence emission up to λ max = 588 nm 13 . CTZ- v has the same effect on photoprotein bioluminescence spectra; obelin activated with CTZ- v (obelin- v ) demonstrates red-shifted bioluminescence with a peak at 532 and a shoulder at 415 nm while obelin with native CTZ emits light with a maximum at 480 nm and a shoulder at 400 nm 14 , 15 .…”

“…Unexpectedly, the structures of obelin- v and obelin bound with native CTZ revealed almost no difference; only minor rearrangement in hydrogen-bond pattern and slightly increased distances between the key active site residues and 2-hydroperoxyCTZ- v were found. Thus, the minor changes in the substrate microenvironment observed in the obelin- v active site were proposed to account for the low efficiency of generating an electronic excited state ( Φ S ) resulting in a low bioluminescence activity of obelin- v 15 .…”

“…Unfortunately, it hinders the usage of CTZ-v analogue as a substrate of the photoprotein bioluminescence. To shed light on the reasons behind the low efficiency of photoprotein bioluminescence with CTZ-v, the crystal structure of Ca 2+ -regulated photoprotein obelin bound with 2-hydroperoxy adduct of CTZ-v (obelin-v) at 1.80 Å resolution was recently solved 15 . This structure provided useful information about coelenterazinebinding cavity of obelin-v before the bioluminescence reaction.…”