Cation-driven Optical Properties of Artificial Luciferases

Abstract

The present study demonstrates cation-driven optical properties of artificial luciferases (ALucs) from copepod luciferases, as an optical readout for bioanalysis. An assignment of the supersecondary structure code (SSC) of ALucs revealed that ALucs carry a helix-loop-helix structure, which appears at the same sites of the EF-hands of typical Ca 2+ -binding proteins. A mutagenesis study shows that the EF-hand-like structure is a pivotal site for enzymatic activity. The effects of 20 kinds of mono-and multivalent cations on ALuc activities were estimated with column-purified ALuc16. High pH values boost the ALuc activities with both the native coelenterazine and an analog called 6-pi-OH-CTZ. Multivalent cations, Ca(II), Mg(II), and Cr(VI), elevate and prolong the ALuc activities, whereas Co(II), Cu(II) and Pb(II) greatly hamper the ALuc activities. Ca(II) greatly prolongs the optical intensities, suggesting a contribution to the structural robustness of ALucs. The inhibitory effect of multivalent cations on the ALuc activities was utilized for creating dose-response curves. The intrinsic cation-driven selectivity and optical intensity of ALucs enable researchers to constitute de novo biosensors for multivalent cations.

Molecular Imaging of Retinoic Acids in Live Cells Using Single-Chain Bioluminescence Probes

“…(ii) The ∼5-fold stronger background intensities of Sara #12 than those of Sara #12ctrl may reflect a basal, nonspecific PPI between the LXXLL motif and the RARα LBD positioned inside Sara #12 in the absence of at-RA. (iii) The basal PPI may happen through nongenomic pathways activated by metabolites in the culture medium or the conformational change induced by the interaction of other cellular proteins, as discussed before; , and (iv) In accordance with the λ max of Sara #12 and Sara #12ctrl, intramolecular complementation indeed occurs between the split N- and C-terminal fragments of ALuc16 inside the probes, considering the λ max of ALuc16 is known to be at ∼500 nm. , …”

“…(iii) The basal PPI may happen through nongenomic pathways activated by metabolites in the culture medium or the conformational change induced by the interaction of other cellular proteins, as discussed before; 27,28 and (iv) In accordance with the λ max of Sara #12 and Sara #12ctrl, intramolecular complementation indeed occurs between the split N-and C-terminal fragments of ALuc16 inside the probes, considering the λ max of ALuc16 is known to be at ∼500 nm. 29,30 Ex Vivo BL Imaging of Tumor Tissues Extracted from Mice Bearing MDA-MB-231 Tumor Xenografts Stably Expressing Sara #12 or Sara #12ctrl. The in vivo imaging of ligand-activated signal would be a useful technique for measuring ligand biodistribution in different organs.…”

Azide- and Dye-Conjugated Coelenterazine Analogues for a Multiplex Molecular Imaging Platform

“…The bioluminescence kinetic properties (K m and V max ) of affinity column-purified ALuc16 according to nCTZ and 6-N 3 -CTZ were determined. The column-purified ALuc16 was prepared with the same method as that of Kim et al 38 Briefly, the cDNA encoding ALuc16 was subcloned into pOPTHM vector and expressed in the bacterial strain Shuffle T7 Express lysS (New England Biolabs). The lysate was purified with HisTrap HP column (GE Healthcare) in an A KTA Purifier system (GE Healthcare).…”