Fig 1.
Luminescence reaction of coelenterazine (CTZ) catalyzed by the CTZ-utilizing luciferase and chemical structures of CTZ analogs and deaza-CTZ analogs.
A. Oxidation process of CTZ with O2 by CTZ-utilizing luciferases and the degradation product of coelenteramine (CTM), 4-hydroxyphenylacetic acid (4HPAA), and 4-hydroxyphenylpyruvic acid (4HPPA) through 2-peroxycoelenterazine (CTZ-OOH). B. Chemical structures of C2- and C6-modified CTZ analogs. The C6-group of CTZ analogs was colored in red, and the C2- and C8-groups of CTZ analogs were colored in blue. C. Chemical structures of deaza-analogs for CTZ and CTZ-OOH as inhibitors.
Fig 2.
Schematic representation of the reverse mutants for nanoKAZ (nK) and their luminescence activities using coelenterazine (CTZ) and bis-coelenterazine (bis-CTZ) as substrates.
A. Chimeric proteins between wild KAZ (wK) and nanoKAZ (nK). wK/nK, 1–82 aa of wK and 83–169 aa of nK; nK/wK, 1–82 aa of nK and 83–169 aa of wK. B. Reverse mutants of wK/nK chimera with a single amino acid substitution at the carboxyl terminal region of nanoKAZ (83–169 aa). C. Reverse mutants of nanoKAZ with three or four amino acid substitutions at the amino- terminal region of nanoKAZ (1–82 aa). D. Reverse mutants of nanoKAZ with a single amino acid substitution at the amino-terminal region of nanoKAZ (1–27 aa). E. Reverse mutants of nanoKAZ with double amino acid substitutions at the amino-terminal region of nanoKAZ (1–27 aa).
Fig 3.
SDS-PAGE analyses of the soluble fractions of the reverse mutants expressed in E. coli cells using a pCold-ZZ-P-X vector (A-E) and the purified nanoKAZ, QL-nanoKAZ, and SNH-nanoKAZ from E. coli cells (F). A-E, the soluble fractions of mutant proteins obtained from E. coli cells by centrifugation at 12,000 × g for 3 min. Panels A-E correspond to those in Fig 2. The soluble fraction (5 μL) corresponded to 10 μL of the cultured cells was applied on a lane. F, purified nanoKAZ (nK), QL-nanoKAZ (QL-nK), and SNH-nanoKAZ (SNH-nK) from E. coli cells using a Ni-chelate column. Each luciferase (10 μg protein) was applied. M, molecular weight markers. The numbers on the left margin represent the molecular weight of marker proteins (TEFCO): Phosphorylase b (97.4 kDa), bovine serum albumin (69.0 kDa), glutamic dehydrogenase (55.0 kDa), lactic dehydrogenase (36.5 kDa), carbonic anhydrase (29.0 kDa), trypsin inhibitor (20.1 kDa), and lysozyme (14.3 kDa).
Table 1.
Luminescence activities of chimeric proteins among wild KAZ, nanoKAZ, and their reverse mutants of nanoKAZ using coelenterazine (CTZ) and CTZ analogs as a substrate.
Table 2.
Luminescence activities of reverse mutants of nanoKAZ using coelenterazine (CTZ) and CTZ analogs as a substrate.
Fig 4.
Luminescence properties of QL-nanoKAZ.
A. Luminescence kinetics of QL-nanoKAZ with CTZ and its analogs as substrates. B. Normalized luminescence spectra of QL-nanoKAZ with CTZ and its analogs, based on the luminescence intensity of QL-nanoKAZ with CTZ. C. Linearity of luminescence intensity (Imax) of QL-nanoKAZ with CTZ, in comparison with nanoKAZ, SNH-nanoKAZ, GLase, and aequorin at the protein concentrations of 0.3 pg to 3 ng (n = 6). Solid and dashed lines represent blank + 3 SD for aequorin and the CTZ-utilizing luciferases, respectively.
Table 3.
Comparison of luminescence activity of QL-nanoKAZ with other CTZ-utilizing luciferases using aequorin as a light standard.
Table 4.
Comparison of gene expression among nanoKAZ, QL-nanoKAZ, SNH-nanoKAZ, and GLase in CHO-K1 cells.
Table 5.
Expression of QL-nanoKAZ in the presence or absence of the secretory signal peptide sequence from Gaussia luciferase (GLsp) in CHO-K1 cells.
Fig 5.
HPLC analyses of the reaction products from coelenterazine (CTZ) catalyzed by the CTZ-utilizing luciferases.
Authentic samples (0.5 μg each) of coelenteramine (CTM), coelenterazine (CTZ), 3-benzyl-5-(4-hydroxyphenyl)pyrazin-2(1H)-one (CTO), coelenteramide (CTMD), and dehydrocoelenterazine (dCTZ). The black and red lines represent the absorbance at 280 and 330 nm, respectively. The amounts of the reaction products from CTZ by nanoKAZ, QL-nanoKAZ, SNH-nanoKAZ, GLase, RLase, and RLase-547 were determined using the predicted products as standard compounds (Table 6).
Table 6.
Reaction products of coelenteramine (CTM) and coelenteramide (CTMD) from coelenterazine (CTZ) by incubation of various CTZ-utilizing luciferases by HPLC analysis.
Table 7.
Substrate specificities and luminescence properties for purified QL-nanoKAZ, nanoKAZ, SNH-nanoKAZ, and native Oplophorus luciferase (OpLase).
Table 8.
Inhibition of luminescence activity of CTZ-utilizing luciferases with deaza-coelenterazine (daCTZ) analogs as inhibitors.
Table 9.
Statistics of data collection and structure refinement.
Fig 6.
Crystal structure of QL-nanoKAZ.
A. Comparison of the crystal structures between nanoKAZ and QL-nanoKAZ. A superimposed structure of QL-nanoKAZ (green, PDB ID: 7VSX) on that of nanoKAZ (yellow, PDB ID: 5B0U). Red color in QL-nanoKAZ and blue color in nanoKAZ indicate the differences in structure at the α3-helices and Tyr 109, respectively. B. The amino acid residues around Tyr 109 in QL-nanoKAZ and nanoKAZ. A cartoon representation of QL-nanoKAZ (green) superimposed on nanoKAZ (yellow) around Tyr 109. Red- and blue colors at the α3-helices and Tyr 109 are from QL-nanoKAZ and nanoKAZ, respectively.
Fig 7.
Comparison of the secondary structures between nanoKAZ and QL-nanoKAZ.
The amino acid sequences of nanoKAZ and QL-nanoKAZ are shown with their positions of the secondary structure, and the letters highlighted in orange indicate the substituted 16 amino acid residues in wild KAZ to prepare reverse mutations of nanoKAZ. The cylinders and arrows indicate the regions of α-helices (yellow, α1–α4) and β-strands (blue, β1–β11), respectively. The green in the cylinder (α3) and the arrows (β6 and β7) in QK-nanoKAZ indicate the structural differences compared to nanoKAZ. Tyr 109 is highlighted in red.