Estimation of the Phospholipase A2 Selectivity on POPC/POPG Membranes Using the Interaction Map

Abstract

The regulation of the activity and selectivity of phospholipase A2 (PLA2), which is capable of cleaving fatty acid in the second position (sn-2) of the phospholipid, is carried out through the membrane-binding and catalytic sites of the enzyme. For hydrolytic activity, PLA2 must first bind to the phospholipid membrane, and the binding efficiency depends on the composition of the membrane. The membrane-binding site of PLA2 is formed by several tens of amino acids and its composition differs from enzyme to enzyme; hydrophobic and positively charged amino acids play a key role in the interaction. In this work, we investigated the interaction of PLA2 from bee venom with phospholipid bilayers of palmitoyl oleoylphosphatidylcholine (POPC) containing different amounts of palmitoyloleoylphosphatidylglycerol (POPG). On the basis of the measurements of the protein intrinsic fluorescence and the anisotropy of the fluorescence of the lipid probe we propose the construction of lipid–protein interaction maps, which reflect both the efficiency of protein binding and changes in the structure of the membrane. These changes cause alterations in the fluorescence anisotropy of the label, which in turn is a measure of the mobility of the lipid environment of the fluorescent probe. Analysis of interaction maps showed that there is a relationship between lipid mobility and enzyme binding efficiency: the optimum interaction of PLA2 with membranes from a POPC/POPG mixture lies in the region of the highest lipid mobility, and not in the region of the highest negative charge. This dependence complements the existing understanding of the process of recognition of the membrane surface by the enzyme and the selection of lipids by the enzyme already bound to the membrane. The proposed mapping method can be extended to other membrane-active proteins.

Bacterial model membranes under the harsh subsurface conditions of Mars

“…Furthermore, it cannot be excluded that the strong coordination of the negatively charged lipid headgroups by Mg 2+ play an additional role in reducing the PLA2's affinity for the membrane by lowering the membrane negative surface density. Indeed, it was reported that the presence of negatively charged lipids enhances the rate of the reaction, 37 favouring the binding of the enzyme to the membrane, which is the initial step of its action mechanism. Finally, it is important to highlight that, even if our CD experiments did not detect any conformational change of PLA2 imposed by Mg 2+ salts, small local conformational changes ( e.g.…”

sPLA2 Wobbles on the Lipid Bilayer between Three Positions, Each Involved in the Hydrolysis Process

“…The method was used several times to trace sPLA2 binding to the bilayer surface [ 33 , 37 ]. In our previous work, we traced changes in bvPLA2 intrinsic fluorescence upon POPC addition [ 15 , 38 ]. It is important that there is no tryptophan residue in the IBS of bvPLA2 ( Figure 3 , Table S1 ) [ 15 ].…”

Spectroscopy Study of Albumin Interaction with Negatively Charged Liposome Membranes: Mutual Structural Effects of the Protein and the Bilayers

“…TMB-PC were prepared. When the ratio of the probe molecules to lipids is 1/4000, no energy transfer is observed between neighboring TMB fluorophores, and the fluorescence anisotropy depends only on the fluorophore mobility [23]. Fluorescence anisotropy of TMB-PC was obtained in a temperature-controlled cell of an F-4000 (Hitachi, Japan) fluorescence spectrometer at 25 • C at λex 475 nm, λem 505 nm.…”