Cysteine-390 is the binding site of luminous substance with symplectin, a photoprotein from Okinawan squid, Symplectoteuthis oualaniensis

Abstract

Symplectin is a photoprotein from a luminous squid, Symplectoteuthis oualaniensis. It has a luminous substrate, dehydrocoelenterazine (DCZ), linked through a thioether bond with a cysteine residue. We have proven the binding site of luminous substrate in symplectin by using an artificial analogue of DCZ, ortho-fluoro-DCZ (F-DCZ). F-DCZsymplectin emitting strong blue light was reconstituted from apo-symplectin and F-DCZ. Proteolytic digestion of the reconstituted F-DCZ-symplectin afforded peptides including C 390 GLK-F-DCZ (amide), which was detected with a house assembled nano-LC-ESI-Q-TOF-MS. The chromo-peptide derived from the F-DCZ-symplectin after luminescence showed the lower molecular mass than that before the luminescence by 12 mass units, corresponding to the loss of one carbon atom upon emitting light. Thus, we have concluded that F-DCZ analogue binds to Cys390 in symplectin so as to emit light.

Suzuki–Miyaura coupling for general synthesis of dehydrocoelenterazine applicable for 6-position analogs directing toward bioluminescence studies

“…35 Besides these applications, fluorinated-analog has been playing important roles to elucidate the molecular mechanism of the bioluminescence of symplectin photoprotein. 22 Many important syntheses of coelenterazine 1 and its analogs have been reported by several research groups including ours. However, most of the synthetic routes are focused on either modifying the aryl group on the 2-position (R 1 ) or introducing only limited diversified groups at the 6-position (R 2 ) and the 8-position (R 3 ) starting from 2-aminopyrazine analogs as shown in Scheme 2.…”

“…20e22 In 2008, the active center cysteine was determined to be 390-Cys in the 501 amino acids of symplectin. 22 Recently, Kuse independently reported that a commercially available photoprotein pholasin, from Pholas dactylus, showed an increase in the bioluminescence by addition of DCL. 23 Synthesis of coelenterazine or dehydrocoelenterazine has been well established since 1990 until recent 2009.…”

Characterizing the Bioluminescence of the Humboldt Squid, Dosidicus gigas (d'Orbigny, 1835): One of the Largest Luminescent Animals in the World

“…In vitro assay for light emission detection. The method to confirm light emission in vitro was adapted from previous methodologies that studied the photoproteins Aequorin and Symplectin (1,(12)(13)(14). The standard light emission assay consisted of the addition of 50 lL of either the crude extract of photophores or a chromatographic fraction whose activity was being tested for light emission, and diluting this in 940 lL Tris-HCl buffer (pH 8.5, 50 mM, 0.4 M NaCl).…”

Chromophores in photoproteins of a glowing squid and mollusk

“…15) Symplectin has eleven cysteines on its amino acid sequence. 16) One of the residues of cysteines was therefore postulated to form the chromophore for the luminescence of symplectin. The structure of the chromophore was then investigated especially focusing on the carbon atom which was bound to the sulfhydryl residue at the active site of symplectin.…”

“…3). 15,16) During these analyses, the most active DCL analog (2,4-diF-DCL) was prepared. The 2,4-difluoro-DCL was then utilized to analyze the stereochemistry of the chromophore.…”