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Characterization of apo and partially saturated states of calerythrin, an EF-hand protein from S. erythraea: A molten globule when deprived of Ca²⁺

¹ Institute of Biotechnology/NMR laboratory, FIN-00014 University of Helsinki, Helsinki, Finland
² Department of Physics, FIN-00014 University of Helsinki, Helsinki, Finland
³ Department of Physical Chemistry 2, Chemical Centre, Lund University, S-22100 Lund, Sweden
⁴ VTT Biotechnology, FIN-02044, VTT, Espoo, Finland

Reprint requests to: Helena Aitio, Institute of Biotechnology/NMR Laboratory, P.O. Box 56, FIN-00014 University of Helsinki, Helsinki, Finland; e-mail: helena.aitio{at}helsinki.fi; fax: 358-9-19159541.

Calerythrin, a four-EF-hand calcium-binding protein from Saccharopolyspora erythraea, exists in an equilibrium between ordered and less ordered states with slow exchange kinetics when deprived of Ca²⁺ and at low temperatures, as observed by NMR. As the temperature is raised, signal dispersion in NMR spectra reduces, and intensity of near-UV CD bands decreases. Yet far-UV CD spectra indicate only a small decrease in the amount of secondary structure, and SAXS data show that no significant change occurs in the overall size and shape of the protein. Thus, at elevated temperatures, the equilibrium is shifted toward a state with characteristics of a molten globule. The fully structured state is reached by Ca²⁺-titration. Calcium first binds cooperatively to the C-terminal sites 3 and 4 and then to the N-terminal site 1, which is paired with an atypical, nonbinding site 2. EF-hand 2 still folds together with the C-terminal half of the protein, as deduced from the order of appearance of backbone amide cross peaks in the NMR spectra of partially Ca²⁺-saturated states.

Keywords: Calcium-binding protein; calerythrin; CD; EF-hand; molten globule; NMR; SAXS

Abbreviations: BSCP, sarcoplasmic calcium-binding protein from Branchiostoma lanceolatum • C-CaVP, C-terminal fragment of calcium vector protein • CD, circular dichroism • DTT, dithiothreitol • EDTA, ethylenediaminetetraacetic acid • EGTA, ethylene glycol-bis(ß-aminoethyl ether)-N,N,N`,N`-tetraacetic acid • HSQC, heteronuclear single quantum coherence • NMR, nuclear magnetic resonance • NSCP, sarcoplasmic calcium-binding protein from Nereis diversicolor • SAXS, small-angle X-ray scattering • SCP, sarcoplasmic calcium-binding protein.

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