The light-sensitive photoprotein berovin from the bioluminescent ctenophore Beroe abyssicola: a novel type of Ca(2+) -regulated photoprotein.
Light-sensitive Ca(2+) -regulated photoproteins are responsible for the bright bioluminescence of ctenophores. Using functional screening, four full-size cDNA genes encoding the same 208-amino-acid polypeptide were isolated from two independent cDNA libraries prepared from two Beroe abyssicola specimens. Sequence analysis revealed three canonical EF-hand calcium-binding sites characteristic of Ca(2+) -regulated photoproteins, but a very low degree of sequence identity (27-29%) with aequorin-type photoproteins, despite functional similarities. Recombinant berovin was expressed in Escherichia coli cells, purified, converted to active photoprotein and characterized. Active berovin has absorption maxima at 280 and 437 nm. The Ca(2+) -discharged protein loses visible absorption, but exhibits a new absorption maximum at 335 nm. The berovin bioluminescence is blue (λ(max) = 491 nm) and a change in pH over the range 6.0-9.5 has no significant effect on the light emission spectrum. By contrast, the fluorescence of Ca(2+) -discharged protein (λ(ex) = 350 nm) is pH sensitive: at neutral pH the maximum is at 420 nm and at alkaline pH there are two maxima at 410 and 485 nm. Like native ctenophore photoproteins, recombinant berovin is also inactivated by light. The Ca(2+) concentration-effect curve is a sigmoid with a slope on a log-log plot of ∼ 2.5. Although this curve for berovin is very similar to those obtained for obelin and aequorin, there are evident distinctions: berovin responds to calcium changes at lower concentrations than jellyfish photoproteins and its Ca(2+) -independent luminescence is low. Recombinant berovin was successfully expressed in mammalian cells, thereby demonstrating potential for monitoring intracellular calcium. Database The nucleotide sequences have been deposited in the GenBankTM/EBI Data Bank with accession numbers: apoberovin cDNA genes, JN673813 (BA1), JN673814 (BA2), JN673815 (BA3), JN673816 (BA4); fragment 18S rRNA, JN673817 (BA-rRNA5).
Photobiology Lab, Institute of Biophysics, Russian Academy of Sciences, Krasnoyarsk, Russia.
Svetlana V
SV
Markova
Ludmila P
LP
Burakova
Stefan
S
Golz
Natalia P
NP
Malikova
Ludmila A
LA
Frank
Eugene S
ES
Vysotski
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000494:pharmacology*; D003001:Cloning, Molecular; D006224:Cricetinae; D037302:Ctenophora / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D008164:Luminescent Proteins; D011994:Recombinant Proteins; D008156:Luciferases; D002118:Calcium