Modulation of the Activity of an Integral Membrane Protein by Phospholipids in Mixed Micelles

Abstract

Although the importance of lipid-protein interactions in determining the biological function of integral membrane proteins is well recognized, the underlying molecular mechanisms remain unclear. In this work we explore the modulation of the enzymatic activity of an integral membrane protein by phospholipids, using the Plasma Membrane Calcium Pump (PMCA) reconstituted in mixed detergent C12E10-phospholipid micelles as model system. Increasing amounts of phospholipids were added to purified PMCA preparations reconstituted in detergent micelles producing a reversible increase in activity reaching a maximum value. No major structural changes occur during this process neither in the protein as assessed by far UV circular dichroism (CD) and tryptophan fluorescence nor in the micellar system as determined by fluorescence spectroscopy and fluorescence correlation spectroscopy (FCS). In addition the relative affinities of phospholipids for the PMCA transmembrane region were evaluated by a FRET method using a pyrene labelled PC as fluorescent probe. These results were analyzed in terms of a macroscopic model that includes the affinities of the phospholipids covering the PMCA transmembrane region and a transduction parameter that correlates the composition of the boundary monolayer with the enzyme activity. The model predictions show good agreement with the experimental data, linking amphiphile/protein interactions with enzymatic activity.