Thermal stability of the plasma membrane calcium pump. Quantitative analysis of its dependence on lipid-protein interactions.
Thermal stability of plasma membrane Ca(2+) pump was systematically studied in three micellar systems of different composition, and related with the interactions amphiphile-protein measured by fluorescence resonance energy transfer. Thermal denaturation was characterized as an irreversible process that is well described by a first order kinetic with an activation energy of 222 +/- 12 kJ/mol in the range 33-45 degrees C. Upon increasing the mole fraction of phospholipid in the mixed micelles where the Ca(2+) pump was reconstituted, the kinetic coefficient for the inactivation process diminished until it reached a constant value, different for each phospholipid species. We propose a model in which thermal stability of the pump depends on the composition of the amphiphile monolayer directly in contact with the transmembrane 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 phospholipid/detergent for the hydrophobic transmembrane surface of the protein (K(LD)) showing that those phospholipids with higher affinity provide greater stability to the Ca(2+) pump. We developed a method for directly measure K(LD) 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 strong evidence to support its validity.
Levi
V
V
Instituto de Química y Fisicoquímica Biológicas-Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956-1113 Buenos Aires, Argentina.
Rossi
J P
JP
Echarte
M M
MM
Castello
P R
PR
González Flecha
F L
FL
D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D027682:Cation Transport Proteins; D004912:Erythrocytes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D010743:Phospholipids / Q000737:chemistry; D053499:Plasma Membrane Calcium-Transporting ATPases; D011092:Polyethylene Glycols / Q000737:chemistry; D011489:Protein Denaturation; D011510:Proteolipids / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013696:Temperature
D027682:Cation Transport Proteins; D010743:Phospholipids; D011510:Proteolipids; C020485:proteoliposomes; D011092:Polyethylene Glycols; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases