title: Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein.
abstract: The full-length cDNA genes encoding five new isoforms of Ca(2+)-regulated photoprotein mitrocomin from a small tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and subsequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed suggestion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30Å resolution which expectedly revealed a high similarity with the structures of other hydromedusan photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473-474nm with no shoulder at 400nm). Fluorescence spectra of Ca(2+)-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca(2+)-discharged aequorin, but different from Ca(2+)-discharged obelins and clytin which fluorescence is red-shifted by 25-30nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specific activity and efficiency of apo-mitrocomin conversion into active photoprotein, in contrast to C-terminal Pro of other photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan photoproteins with different bioluminescence properties.
authors.affiliation: Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia.
authors.forename: Ludmila P
authors.initials: LP
authors.lastname: Burakova
authors.affiliation: Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia; National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Pavel V
authors.initials: PV
authors.lastname: Natashin
authors.affiliation: Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia.
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Markova
authors.affiliation: Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Eremeeva
authors.affiliation: Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia.
authors.forename: Natalia P
authors.initials: NP
authors.lastname: Malikova
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Chongyun
authors.initials: C
authors.lastname: Cheng
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650500, China; iHuman Institute, ShanghaiTech University, Shanghai 201210, China. Electronic address: zjliu@ibp.ac.cn.
authors.forename: Zhi-Jie
authors.initials: ZJ
authors.lastname: Liu
authors.affiliation: Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia. Electronic address: eugene.vysotski@gmail.com.
authors.forename: Eugene S
authors.initials: ES
authors.lastname: Vysotski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D049449:Luminescence; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D017384:Sequence Deletion; D014443:Tyrosine
chemical_list: D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; C083952:mitrocomin protein, Mitrocoma celluria; D014443:Tyrosine; D002118:Calcium