Conformation and thermal denaturation of apocalmodulin: role of electrostatic mutations.
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.
Protasevich
I
I
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
Ranjbar
B
B
Lobachov
V
V
Makarov
A
A
Gilli
R
R
Briand
C
C
Lafitte
D
D
Haiech
J
J
D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D055672:Static Electricity
D002147:Calmodulin; D002118:Calcium