Effects of trifluoperazine on calcium binding by calmodulin. Heat capacity and entropy changes.

M Tanokura,K Yamada

doi:10.1016/s0021-9258(18)67449-6

Abstract

The possible structural changes of the calmodulin-trifluoperazine (TFP) complex caused by Ca2+ binding have been analyzed by microcalorimetric titrations. Titrations of calmodulin with Ca2+ in the presence of 8-fold molar excess TFP have been made both in the absence and presence of Mg2+, at pH 7.0, and at 5, 15, and 25 degrees C. At high concentrations of TFP calmodulin forms a complex with TFP even in the absence of Ca2+. The reaction of the calmodulin-TFP complex with Ca2+ is exothermic, both in the presence and absence of Mg2+. In the presence of Mg2+ the reaction is driven almost entirely by a favorable enthalpy change. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of this complex on Ca2+ binding have been estimated by the empirical method of Sturtevant (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240). In the presence of Mg2+, the vibrational as well as hydrophobic entropy is slightly increased in a parallel manner by Ca2+ binding to each of the binding sites. In contrast, when Mg2+ is absent, the hydrophobic entropy gradually increases on Ca2+ binding, but the vibrational entropy decreases. These changes of entropy indicate the assembling of non-polar groups on the surface of the complex and suggest that the overall structure is loosened in the presence of Mg2+, but tightened in the absence of Mg2+.

Highlights

The possible structural changes of the calmodulin- rimetric titrations indicatedthat calmodulin andTFP form a trifluoperazine (TFP) complex caused by Ca2+binding complex even in the absence of Ca2+at high concentrations have been analyzed by microcalorimetric titrations. of T F P, which is in agreement with other reports describing
The hydrophobic and vibrational contributions to the heat capacity and entropy changes of t h e estimated by the empirical method ofSturtevant
A pure calmodulin fraction, phobic entropy gradually increases on Caz+binding, but the vibrational entropy decreases. These changes of entropy indicate the assembling of non-polar groups on the surface of the complex and suggest that the confirmed by polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate [18,19,20], was collected, precipitated with 5% trichloroacetic acid, dissolved in a small volume of 1 M Tris, and dialyzed against 1 mM NaHC03as described previously [7]

Summary

Masaru Tanokura and Kazuhiro Yamada

A pure calmodulin fraction, phobic entropy gradually increases on Caz+binding, but the vibrational entropy decreases These changes of entropy indicate the assembling of non-polar groups on the surface of the complex and suggest that the confirmed by polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate [18,19,20], was collected, precipitated with 5% trichloroacetic acid, dissolved in a small volume of 1 M Tris, and dialyzed against 1 mM NaHC03as described previously [7]. To obtain the heat attributable to Ca2+or Mg2+ binding in the presence of TFP, the observed heat must be corrected for the heat conformational changes, we have previously examined possi- caused by the interaction between the Pipes buffer and protons ble conformational changes associated with Ca2+binding to released The latter was determined by measuring the amount of calmodulin in the absence and presence of TFP [6,7,8]. The mole fraction sites (f) were somewhat smaller than 1.0 for almost all the measurements, but similar results

RESULTS

Temperature f logK

The changes in heat capacity and entroapssyociated solely

Ca binding site