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Evidence of Noncovalent Dimerization of Calmodulin

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Evidence of Noncovalent Dimerization of Calmodulin Eur. J. Biochem. 261, 337±344 (1999) q FEBS 1999 Evidence of noncovalent dimerization of calmodulin Daniel Lafitte1, Albert J. R. Heck1, Tessa J. Hill1, Kornelia Jumel2, Stephen E. Harding2 and Peter J. Derrick1 1Department of Chemistry, University of Warwick, Coventry, UK; 2NCMH Unit, School of Biological Sciences, University of Nottingham, Sutton Bonington, UK Calcium-binding proteins, such as S-100, dimerize readily, and this phenomenon 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; Kilby, P.M., Van Eldik, L.J. & Roberts, G.C. (1996) Structure 4, 1041±1052]. We have investigated by Fourier-transform ion cyclotron resonance (FTICR) MS the conformational states of the calcium-binding protein calmodulin, and present clear evidence for a calmodulin dimer formed as a result of noncovalent interactions between folded monomers. Ultra-high-resolution electrospray 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 calmodulin was electrosprayed from buffer, monomers ions with only 5±10 charges were detected. CD measurements for calmodulin in buffered solution revealed that its a-helical content was significantly higher than that for calmodulin in acetonitrile/water solutions, consistent with a proposition that changes in charge state distributions observed in the MS experiments reflect differing states of calmodulin folding. Under buffered conditions, noncovalently bound calmodulin dimers were observed by ESI FTICR MS. Analytical ultracentrifugation experiments carried out in the same solution conditions as those used in the MS experiments were consistent with the proposed calmodulin dimer±monomer equilibrium. The ultra-high mass resolution achieved with the 9.4 T FTICR mass spectrometer allowed unequivocal identification of the noncovalent, as opposed to covalent, character of the calmodulin dimer. Keywords: calmodulin; dimer; electrospray ionization; Fourier-transform ion cyclotron resonance (FTICR); noncovalent interactions. An understanding of protein conformational changes is especi- protein conformation and noncovalent protein±ligand binding in ally important with a protein such as calmodulin that is able to solution [17,18]. Fourier-transform ion cyclotron resonance activate many different target enzymes [1,2]. Dimerization (FTICR) MS is important in this context because of the processes, like conformational changes, may modify the inter- exceptionally high mass resolution accessible (see [19] for a faces available for target protein recognition, which can, in turn, review). High-field FTICR allows mass measurements with either activate or inhibit a target protein's action upon binding mass resolutions of more than 100 000 and mass accuracies of [3,4]. There is a growing need to develop methods to charac- better than one part per million, and, as a consequence of terize protein±protein interactions. To date sedimentation allowing the resolution of the isotopic components of a given equilibrium [5], X-ray crystallography [6], NMR [7] and X-ray species, brings a special advantage to studies of proteins and and light scattering [8] have appeared to be the established other macromolecules. methods for characterizing dimeric motifs. Nevertheless, dimer The superfamily of calcium-binding proteins is an illustrative characterization remains quite difficult, especially when involv- example of how protein conformational changes serve to ing weak noncovalent interactions. transduce external stimuli into a cellular response. As a result In recent years, electrospray ionization (ESI) MS has become of calcium binding, calcium-binding proteins undergo confor- important in structure±activity relationship studies. This tech- mational changes [20,21] leading specifically to conformations nique allows proteins with molecular masses even greater than that activate the appropriate enzymes. Calmodulin is one of the 100 kDa to be transported from their natural aqueous environ- more representative proteins of this family (for reviews see ments into the gas phase with, under appropriate conditions, the [22,23]). retention of primary and even higher-order structures [9±11]. It After calcium activation [24,25], calmodulin is able to inter- has been argued that in vacuo ESI mass spectra may reflect act with a range of enzymes which include myosin light chain aqueous solution chemistry [12±16], and as a consequence ESI kinase, fodrin, calmodulin kinase II, phosphodiesterase and MS is beginning to be used quite widely to gain information on inositol 1,4,5-trisphosphate kinase 1. These enzymes are known to be involved in many physiological processes of importance, Correspondence to P. J. Derrick, Department of Chemistry, such as glycolysis, muscle contraction, cell division, cell growth, University of Warwick, Coventry CV4 7AL, UK. Fax: +44 1203 523819, exocytosis and endocytosis. In addition, calmodulin may interact Tel.: +44 1203 523818, E-mail: [email protected] without calcium activation as is the case with neuromodulin and Abbreviations: ESI, electrospray ionization; FTICR, Fourier-transform ion brush-border myosin I [26]. cyclotron resonance. The structure of the calmodulin±calcium complex has been (Received 19 November 1998; revised 4 January 1999; accepted resolved by X-ray crystallography [27±29] and NMR studies 28 January 1999) [30,31], and calmodulin is shown to exhibit a dumbbell shape 338 D. Lafitte et al.(Eur. J. Biochem. 261) q FEBS 1999 composed of two globular domains linked by a long flexible to facilitate the desolvation of the droplets formed at the begin- central helix [32]. Each globular domain contains two helix± ning of the electrospray process. Such solutions were found to loop±helix motifs or EF-hands which are expected to be the be optimal for producing stable electrospray conditions, but of main calcium-binding sites [32,33]. Previous biophysical studies course do not mimic physiological conditions. To represent have shown that additional sites of weaker affinity are also present physiological conditions more closely, electrospray solutions on calmodulin [34,35]. The structure of the bare calmodulin were prepared in 10 mm ammonium acetate (using high-purity protein without calcium (apo-calmodulin) proved more difficult to water) and buffered to the appropriate pH using acetic acid or elucidate and this was achieved, only a few years ago, by NMR ammonia. The concentration of calmodulin was 50 mm in all [26,36,37]. X-ray-crystallographic data on apo-calmodulin protein experiments, unless stated otherwise. are, as far as we are aware, not yet available. Calmodulin is a highly flexible protein and can transitorily CD measurements adopt a more extended conformation, which exposes highly CD spectra were recorded on a Jasco J-715 spectropolarimeter, hydrophobic regions. This feature is common among the calcium- and corrected for background CD absorbance using the water/ binding protein superfamily and it has been suggested that these acetonitrile and ammonium acetate solutions. Concentrations of hydrophobic regions play a role in the dimerization of some calmodulin in the solutions were determined by UV absorbance calcium-binding proteins, e.g. troponin C [5]. to allow accurate determination of the a-helical content. We present here a study of dimerization of calmodulin using ESI coupled with FTICR MS. CD and ultracentrifugation experi- Ultracentrifugation analysis ments were performed using the same sample conditions as for MS. Sedimentation equilibrium experiments of the `low-speed' type [43] were performed using a Beckman Optima XL-I analytical ultracentrifuge fitted with absorption and Rayleigh interference EXPERIMENTAL PROCEDURES optics together with full on-line computer data capture and analysis facilities (Beckman, Palo Alto, CA, USA) [44]. Double- Protein synthesis and purification sector cells of 12-mm optical path length were used with a DNA-encoded calmodulin was produced by previously described solution column of <1 mm (80 mL) and run at 20 8Cata procedures and purified by column chromatography [38,39]. rotor speed of 130 000 g. A partial specific volume of The purity of the protein was checked by SDS/PAGE and high- 0.7235 mL´g21 was used, calculated from the amino acid pressure capillary electrophoresis and was found to be around sequence according to the conserved formula of Perkins [45]. 99%. In all experiments ultrapure water (Elga system) and Equilibrium solute distributions were analysed using MSTARA plastic ware, washed in 1 m HCl, were used to minimize metal (for absorption optics) and MSTARI (for interference optics) cation and other contamination. Calmodulin (4.5 mg) was [44] which use the computerized M* method [46,47]. dissolved in 2.5 mL of ammonium acetate (10 mm, pH 5.9) and purified over a desalting PD10 column (Pharmacia, Uppsala, RESULTS AND DISCUSSION Sweden), previously equilibrated with ammonium acetate. Approximately 10 fractions, of 1 mL each, were collected. `Denaturing' conditions Bradford reagent was used to check for the presence of the Figure 1A shows the ESI FTICR mass spectrum obtained for protein in each individual fraction, and showed that the last calmodulin in a 1 : 1 water/acetonitrile solution containing 1% seven fractions contained protein.
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