Purification of QL-nanoKAZ from 800 mL of cultured <i>E</i>. <i>coli</i> cells using a Ni-chelate column.

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S1 Raw images -

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Expression of QL-nanoKAZ in the presence or absence of the secretory signal peptide sequence from <i>Gaussia</i> luciferase (GLsp) in CHO-K1 cells.

Expression of QL-nanoKAZ in the presence or absence of the secretory signal peptide sequence from Gaussia luciferase (GLsp) in CHO-K1 cells.</p

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.</p

Inhibition of luminescence activity of CTZ-utilizing luciferases with deaza-coelenterazine (daCTZ) analogs as inhibitors.

Inhibition of luminescence activity of CTZ-utilizing luciferases with deaza-coelenterazine (daCTZ) analogs as inhibitors.</p

Statistics of data collection and structure refinement.

Statistics of data collection and structure refinement.</p

Primer list used for site-directed mutagenesis to prepare reverse mutant genes for nanoKAZ by PCR.

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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.</p

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.</p

Reaction products of coelenteramine (CTM) and coelenteramide (CTMD) from coelenterazine (CTZ) by incubation of various CTZ-utilizing luciferases by HPLC analysis.

Reaction products of coelenteramine (CTM) and coelenteramide (CTMD) from coelenterazine (CTZ) by incubation of various CTZ-utilizing luciferases by HPLC analysis.</p