Fragments from actin binding protein (ABP-280; filamin) insert into reconstituted lipid layers

Previous computer analyses suggested two possible lipid binding sites, resi dues 49-71 and 131-155, of the primary amino acid sequence on ABP-280 (fila min), which could facilitate membrane attachment/insertion. We expressed th ese regions as fusion proteins with schistosomal GST and investigated their interaction with mixtures of zwitterionic (dimyristoyl-L-alpha-phosphatidy lcholine, DMPC) and anionic (dimyristoyl-L-alpha-phosphatidylglycerol, DMPG ) phospholipids in reconstituted lipid bilayers by differential scanning ca lorimetry (DSC), Using vesicles of mixed DMPC/ DMPG with increasing fusion protein concentrations, we established in calorimetric assays a decrease of the main chain transition enthalpy, Delta H, and a shift in chain melting temperature. This is indicative of the insertion of these fragments into th e hydrophobic region of lipid membranes. We confirmed these findings by the film balance technique using lipid monolayers (DMPG). The binding judged f rom both methods was of moderate affinity, (C) 1999 Academic Press.