ALBERT

Description: Ca2+-regulated photoproteins responsible for bioluminescence of a variety of marine organisms are single-chain globular proteins within the inner cavity of which the oxygenated coelenterazine, 2-hydroperoxycoelenterazine, is tightly bound. Alongside with native coelenterazine, photoproteins can also use its synthetic analogues as substrates to produce flash-type bioluminescence. However, information on the effect of modifications of various groups of coelenterazine and amino acid environment of the protein active site on the bioluminescent properties of the corresponding semi-synthetic photoproteins is fragmentary and often controversial. In this paper, we investigated the specific bioluminescence activity, light emission spectra, stopped-flow kinetics and sensitivity to calcium of the semi-synthetic aequorins and obelins activated by novel coelenterazine analogues and the recently reported coelenterazine derivatives. Several semi-synthetic photoproteins activated by the studied coelenterazine analogues displayed sufficient bioluminescence activities accompanied by various changes in the spectral and kinetic properties as well as in calcium sensitivity. The poor activity of certain semi-synthetic photoproteins might be attributed to instability of some coelenterazine analogues in solution and low efficiency of 2-hydroperoxy adduct formation. In most cases, semi-synthetic obelins and aequorins displayed different properties upon being activated by the same coelenterazine analogue. The results indicated that the OH-group at the C-6 phenyl ring of coelenterazine is important for the photoprotein bioluminescence and that the hydrogen-bond network around the substituent in position 6 of the imidazopyrazinone core could be the reason of different bioluminescence activities of aequorin and obelin with certain coelenterazine analogues.

Description: Bioluminescent proteins are widely used as reporter molecules in various in vitro and in vivo assays. The smallest isoform of Metridia luciferase (MLuc7) is a highly active, naturally secreted enzyme which, along with other luciferase isoforms, is responsible for the bright bioluminescence of marine copepod Metridia longa. In this study, we report the construction of two variants of a hybrid protein consisting of MLuc7 and 14D5a single-chain antibody to the surface glycoprotein E of tick-borne encephalitis virus as a model fusion partner. We demonstrate that, whereas fusion of a single-chain antibody to either N- or C-terminus of MLuc7 does not affect its bioluminescence properties, the binding site on the single-chain antibody influences its binding capacity. The affinity of 14D5a-MLuc7 hybrid protein (KD = 36.2 nM) where the C-terminus of the single-chain antibody was fused to the N-terminus of MLuc7, appeared to be 2.5-fold higher than that of the reverse, MLuc7-14D5a (KD = 87.6 nM). The detection limit of 14D5a-MLuc7 hybrid protein was estimated to be 45 pg of the recombinant glycoprotein E. Although the smallest isoform of M. longa luciferase was tested as a fusion partner only with a single-chain antibody, it is reasonable to suppose that MLuc7 can also be successfully used as a partner for genetic fusion with other proteins.

Description: Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+-dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP-aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca2+ sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty-four amino acid positions in and around EF-hand Ca2+-binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C-terminal tail, which exhibited markedly higher Ca2+ sensitivity than wild-type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca2+ sensitivity than wild-type Redquorin, and four of them (RedquorinXS) matched the Ca2+ sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt-Redquorin, and one, RedquorinXS-Q159T, outperformed GA. Finally, wide-field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS-Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg2+. Our results indicate that RedquorinXS-Q159T is a red light-emitting Ca2+ sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice.