Conformation and Thermal Denaturation of Apocalmodulin:  Role of Electrostatic Mutations

Abstract

Scanning microcalorimetry and circular dichroism were used to study conformational state and heat denaturation of Ca2+-free synthetic calmodulin (SynCaM) and three charge reversal mutants. We produced evidence for the major role of the electrostatic potential in the stability and flexibility of SynCaM. The substitution of 118DEE120 by 118KKK120 (SynCaM12A) does not influence the flexibility of the protein; the replacement of 82EEE84 by 82KKK84 (SynCaM8) decreases its level, while the combination of these two mutations in SynCaM18A significantly increases the flexibility. The heat denaturation of apoSynCaM and its mutants is well approximated by two two-state transitions with the lower-temperature transition corresponding to C-terminal lobe melting and the higher-temperature one to N-terminal lobe melting. The difference in transition temperatures for the two lobes decreases in SynCaM8 and increases in SynCaM18A, suggesting a modification in the influence of one lobe to the other. The electrostatic mutations change the parameters of thermal denaturation of SynCaM lobes in a similar way as pH conditions affect thermal transition parameters of multidomain proteins, leading to a linear temperature dependence of transition enthalpy. One domain of the N-terminal lobe in apoSynCaM18A is unfolded in the native state. Near-UV CD spectra point out the invariability of the local structure of aromatic residues upon mutations, although the secondary structure undergoes striking transformations. Cacodylate ions strongly and specifically alter the helical content of SynCaM. Our data unambiguously demonstrate that the two lobes are not independent, and interactions between the lobes are mediated by the electrostatic potential of the molecule.

Energetics of Target Peptide Binding by Calmodulin Reveals Different Modes of Binding

“…It was found that the stoichiometry of CaMKI/Ca 2ϩ -CaM complex formation in sodium cacodylate buffer is on the order of 0.8:1. Somewhat peculiar effects of cacodylate on the conformation and stability of calmodulin have also been previously observed (54).…”

Apocalmodulin Binds to the Myosin Light Chain Kinase Calmodulin Target Site

“…The reversibility of unfolding was checked routinely by sample reheating after cooling in the calorimetric cell. The calorimetric denaturation enthalpy (⌬H cal ) and the partial molar heat capacity of the protein (C p ) were determined as described elsewhere (13), assuming that the molecular mass of SynCaM is 16628 Da and the partial specific volume is 0.72 cm 3 g Ϫ1 (7). To analyze functions of excess heat capacity, the MicroCal Origin software was used.…”

“…There was no ITC signal upon apoSynCaM titration with EGTA. Cacodylate buffer (10 mM) was used because we have shown that this buffer concentration does not induce a major change in the secondary structure of apoprotein (7). Some experiments were done in 50 mM Hepes.…”

“…The characterization of this overall system in thermodynamic terms requires investigation of the apoCaM thermodynamic state in the absence or presence of an interacting peptide. Concerning CaM, we have shown that a combination of isothermal titration calorimetry and differential scanning calorimetry allows one to pinpoint the thermodynamic properties of this exquisitely tuned system (7,8).…”

A stopped-flow fluorescence study of the native and modified lysozyme

“…The instrument was calibrated with (+)-10-camphorsulfonic acid, assuming [θ]291 = 7820 deg cm 2 dmol −1 (Schippers & Deckers 1981) and with JASCO standard non-hydroscopic ammonium (+)-10-camphorsulfonate, assuming [θ]290.5 = 7910 deg cm 2 dmol −1 (Takakuwa et al 1985). Noise in the data was smoothed using the JASCO J-715 software, including the fast Fourier-transform noise reduction routine which allows the enhancement of most noisy spectra without distorting their peak shapes (Protasevich et al 1997). …”