Thermal Stability of the Plasma Membrane Calcium Pump. Quantitative Analysis of Its Dependence on Lipid-Protein Interactions

Abstract

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.

Kinetic stability of membrane proteins

“…In addition, this limit value was similar to that obtained for the membrane protein reconstituted in liposomes with the same lipid composition (Levi et al 2000), but lower than that measured for the enzyme in the native membrane. The value characterizing maximal stability (k min ) was found to be dependent on the type of phospholipids included in the mixed micelles, and was also affected when phospholipids undergo chemical modifications, such as nonenzymatic glycation of phosphatidylethanolamine, a common alteration in uncontrolled diabetes mellitus (Levi et al 2008).…”

“…The influence of the relative concentration of phospholipid and detergent on the thermal stability of membrane proteins can be studied using two alternative experimental designs: either the concentration of detergent is kept constant while the amount of added phospholipids is increased, or vice versa. Comparison between these kinds of experiments performed using the plasma membrane calcium pump as a model, showed that similar values of k app were obtained when the mole fractions of the phospholipids in the micelles were similar, even if the phospholipid concentrations in the whole system were different (Levi et al 2000). This indicates that the effect of phospholipids and detergent on membrane protein stability is related to the micellar phase composition but not to the concentration of micelles in the system.…”

“…Levi et al (2000) demonstrate that k app increases steeply when X det,mic approaches 1 (θ PL → 0) and becomes minimal when X PL,mic asymptotically approaches 1. This dependence can be described by a hyperbolic function of θ PL :…”

“…This was the case of several P-ATPases for which the kinetic coefficients describing the time course of the structural changes detected by Trp, ANS and eosin fluorescence, circular dichroism, and ion occlusion capacity are not statistically different from that corresponding to the inactivation process (Cattoni et al 2008;Kaufman et al 2012;Levi et al 2000;Placenti et al 2017), indicating that changes in the enzyme activity and secondary and tertiary structure are all part of the same global change produced by incubation at high temperatures.…”

“…In P-type ATPases, it was found that the decrease in enzyme activity is paralleled by large changes in secondary and tertiary structure (Cattoni et al 2008;Kaufman et al 2012;Levi et al 2000;Placenti et al 2017). The irreversible loss of ATPase activity was not due to either degradation or formation of SDS-stable aggregates of the protein.…”