INSERTION OF FILAMIN INTO LIPID-MEMBRANES EXAMINED BY CALORIMETRY, THE FILM BALANCE TECHNIQUE, AND LIPID PHOTOLABELING

The interaction of the actin-binding protein filamin with mixtures of zwitterionic and anionic phospholipids (DMPC, DMPG, PC, PS) was studie d in reconstituted lipid monolayers and bilayers. Protein-lipid intera ctions were investigated by differential scanning calorimetry, the fil m balance technique, and hydrophobic photoradiolabeling. For calorimet ric assays, multilamellar vesicles (MLVs) and large unilamellar vesicl es produced by the extrusion technique (LUVETs) were used. With MLVs, filamin induced a pronounced drop in phase transition cooperativity. M ixed DMPC/DMPG LUVETs showed a linear decrease of the main phase trans ition enthalpy and a significant shift in temperature for the solidus and liquidus lines with increasing mole fractions of reconstituted fil amin. The insertion of native filamin into uncharged and negatively ch arged lipid monolayers was measured in time/area diagrams with the fil m balance technique. Finally, we have newly synthesized a highly sensi tive lipid analogue, [(125)]TID-PC/16, which selectively labels membra ne-embedded hydrophobic domains of proteins, and which proved to label filamin, supporting evidence that this protein partially inserts into the hydrophobic domain of liposomes.