Obelin mutant with increased sensitivity to calcium.

Authors

Natalia P Malikova, Elena V Eremeeva, Mariya O Vyatkina, Ludmila P Burakova, Igor A Korotov, Svetlana V Markova, Eugene S Vysotski

Published in

International journal of biological macromolecules. Pages 154097. Aug 17, 2026. Epub Aug 17, 2026.

Abstract

For years, Ca²⁺-regulated photoproteins have been successfully used to detect calcium ions, ubiquitous eukaryotic intracellular messengers. Here we report the I144L obelin mutant with dramatically enhanced light output at low [Ca²⁺] which was unexpectedly found among the nineteen mutants with substitution of this conservative residue to all possible amino acids for all-round characterization. The mutant retains high specific activity, light signal kinetics, and spectral properties of wild-type obelin. Moreover, magnesium ions at physiological concentration of 1 mM do not significantly influence its bioluminescence activity. The OL I144L successfully demonstrated its potential as intracellular calcium reporter through assaying endogenous P2Y₂ purinoreceptor activation in CHO cells by displaying a 2.4-fold higher sensitivity to calcium as compared to that of the wild-type obelin. The corresponding mutation was also introduced into the sequences of aequorin and clytin. Both mutants showed the enhanced bioluminescence activity at low [Ca²⁺] as compared to the corresponding wild-type photoproteins, but the increase of light signals in those was significantly lower than that of OL I144L mutant. Based on obelin structure, mutant properties, and the results obtained for other EF-hand Ca²⁺-binding proteins we speculate that the appearance of Leu instead of Ile affects the position of substrate within the inner cavity owing to a little different distances between Leu side chain and 2-hydroperoxycoelenterazine atoms which in turn may slightly shift the α-helices relative to each other changing the angles between them, thereby generating the 'pre-formed' EF-hand structures with a high affinity to calcium.

PMID:
42607904
Bibliographic data and abstract were imported from PubMed on 18 Aug 2026.

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