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

摘要

Symplectin is a photoprotein from a luminous squid, Symplectoteuthisoualaniensis. It has a luminous substrate, dehydrocoelenterazine (DCZ), linked through athioether bond with a cysteine residue. We have proven the binding site of luminous substrate insymplectin by using an artificial analogue of DCZ, ortho-fluoro-DCZ (F-DCZ). F-DCZ-symplectin emitting strong blue light was reconstituted from apo-symplectin and F-DCZ.Proteolytic digestion of the reconstituted F-DCZ-symplectin afforded peptides includingC~(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 thelower molecular mass than that before the luminescence by 12 mass units, corresponding to theloss of one carbon atom upon emitting light. Thus, we have concluded that F-DCZ analoguebinds to Cys390 in symplectin so as to emit light.