Clytin-II (CL-II) is an isotype of the calcium-binding photoprotein clytin-I (CL-I) from Clytia gregaria. CL-II shows approximately 4.5-fold higher initial luminescence intensity than CL-I with the same luminescence capacity, and is a potential candidate for a G-protein-coupled receptor assay among photoproteins. To investigate the high initial luminescence intensity of the chimeric proteins between CL-I and CL-II were prepared and the responsible amino acid residues in CL-II were identified by site-specific mutagenesis of CL-I. The luminescence properties of CL-I were converted to those of CL-II by the replacement of only four amino acids in CL-I, and these amino acids did not interact with 2-peroxycolenterazine. (C) 2015 Elsevier Inc. All rights reserved.