Evidence of noncovalent dimerization of calmodulin

Calcium-binding proteins, such as S-100, dimerize readily, and this phenome non plays an important role in their regulation of target enzymes [Krebs, J ., Quadroni, M. & Van Eldik, L,J. (1995) Nat. Struct. Biol. 2, 711-714; Kil by, P.M., Van Eldik, L.J. & Roberts, G.C. (1996) Structure 4, 1041-1052]. W e have investigated by Fourier-transform ion cyclotron resonance (FTICR) MS the conformational stares of the calcium-binding protein calmodulin, and p resent clear evidence for a calmodulin dimer formed as a result of noncoval ent interactions between folded monomers. Ultra-high-resolution electrospra y ionization (ESI) mass spectra for calmodulin, obtained with a 9.4 T FTICR mass spectrometer, are presented. With the use of denaturing solutions (1 : 1 acetonitrile/water + 1% formic acid), relatively high charge states (20 < z < 10) of monomeric calmodulin ions were detected, whereas when calmodu lin was electrosprayed from buffer, monomers ions with only 5-10 charges we re detected. CD measurements for calmodulin in buffered solution revealed t hat its alpha-helical content was significantly higher than that for calmod ulin in acetonitrile/water solutions, consistent with a proposition that ch anges in charge state distributions observed in the MS experiments reflect differing states of calmodulin folding. Under buffered conditions, noncovalently bound calmodulin dimers were obser ved by ESI FTICR MS. Analytical ultracentrifugation experiments carried out in the same solution conditions as those used in the MS experiments were c onsistent with the proposed calmodulin dimer-monomer equilibrium. The ultra -high mass resolution achieved with the 9.4 T FTICR mass spectrometer allow ed unequivocal identification of the noncovalent, as opposed to covalent, c haracter of the calmodulin dimer.