Fragments from alpha-actinin insert into reconstituted lipid bilayers

Recent experiments have indicated that alpha-actinin interacts with phospho lipid membranes. Using computer analysis methods we determined two possible lipid binding sites capable of membrane attachment/insertion, residues 281 -300 and 720-739 of the primary amino acid sequence on smooth muscle cu-act inin. Having expressed these regions as fusion proteins with schistosomal G ST (glutathione S-transferase), we used differential scanning calorimetry ( DSC) to investigate their interaction with mixtures of zwitterionic (dimyri stoyl-L-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-L-alpha-p hosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers. C alorimetric measurements showed that as fusion protein concentration increa sed, the main chain transition enthalpy decreased and chain melting tempera tures shifted, which is indicative of partial protein insertion into the hy drophobic region of the lipid membranes. Centrifugation assay and subsequen t SDS/Page chromatography confirmed this finding. (C) 1999 Academic Press.