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Microcalorimetric Investigation of the Interactions in the Ternary Complex Calmodulin-Calcium-Melittin

Published inJournal of Biological Chemistry, vol. 262, no. 6, p. 2746-2749
Publication date1987
Abstract

Flow microcalorimetric titrations of calmodulin with melittin at 25 °C revealed that the formation of the high-affinity one-to-one complex in the presence of Ca²⁺ (Comte, M., Maulet, Y., and Cox, J.A. (1983) Biochem. J. 209, 269-272) is entirely entropy driven (ΔH° = 30.3 kJ.mol⁻¹; ΔS° = 275 J.K⁻¹.mol⁻¹). Neither the proton nor the Mg²⁺ concentrations have any significant effect on the strength of the complex. In the absence of Ca²⁺, a nonspecific calmodulin-(melittin)n complex is formed; the latter is predominantly entropy driven, accompanied by a significant uptake of protons and fully antagonized by Mg²⁺. Enthalpy titrations of metal-free calmodulin with Ca²⁺ in the presence of an equimolar amount of melittin were carried out at pH 7.0 in two buffers of different protonation enthalpy. The enthalpy and proton release profiles indicate that: 1) protons, absorbed by the nonspecific calmodulinmelittin complex, are released upon binding of the first Ca²⁺; 2 ) Ca²⁺ binding to the high-affinity configuration of the calmodulin-melittin complex displays an affinity constant ≥10⁷ M⁻¹, i.e. 2 orders of magnitude higher than that of free calmodulin; 3) the latter is even more entropy driven (ΔH° = 7.2 kJ.site⁻¹; ΔS° = 158 J.K⁻¹.site⁻¹) than binding to free calmodulin (ΔH° = 4.7 kJ.site⁻¹; ΔS° = 112 J.K⁻¹.site⁻¹), thus underlining the importance of hydrophobic forces in the free energy coupling involved in the ternary complex.

Funding
  • Swiss National Science Foundation - 3.161.0.85
Citation (ISO format)
MILOS, Mladen et al. Microcalorimetric Investigation of the Interactions in the Ternary Complex Calmodulin-Calcium-Melittin. In: Journal of Biological Chemistry, 1987, vol. 262, n° 6, p. 2746–2749. doi: 10.1016/S0021-9258(18)61569-8
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ISSN of the journal0021-9258
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