Thermal stability of the plasma membrane calcium pump. Quantitative analysis of its dependence on lipid-protein interactions

Thermal stability of plasma membrane Ca2+ pump was systematically studied i n three micellar systems of different composition, and related with the int eractions amphiphile-protein measured by fluorescence resonance energy tran sfer. Thermal denaturation was characterized as an irreversible process tha t is well described by a first order kinetic with an activation energy of 2 22 +/- 12 kJ/mol in the range 33-45 degrees C. Upon increasing the mole fra ction of phospholipid in the mixed micelles where the Ca2+ pump was reconst ituted, the kinetic coefficient fur the inactivation process diminished unt il it reached a constant value, different for each phospholipid species. We propose a model in which thermal stability of the pump depends on the comp osition of the amphiphile monolayer directly in contact with the transmembr ane protein surface. Application of this model shows that the maximal pump stability is attained when 80% of this surface is covered by phospholipids. This analysis provides an indirect measure of the relative affinity phosph olipid/detergent for the hydrophobic transmembrane surface of the protein ( K-LD) showing that those phospholipids with higher affinity provide greater stability to the Ca2+ pump. We developed a method for directly measure K(L D)by using fluorescence resonance energy transfer from the membrane protein tryptophan residues to a pyrene-labeled phospholipid. K-LD values obtained by this procedure agree with those obtained from the model, providing a st rong evidence to support its validity.