Stability of annexin v in ternary complexes with ca(2+) and anionic phospholipids: IR studies of monolayer and bulk phases

Annexin V (AxV) is a member of a family of proteins that exhibit functional ly relevant Ca2+-dependent binding to anionic phospholipid membranes. Prote in structure and stability as a function of Ca2+ and phospholipids was stud ied by bulk phase infrared (IR) spectroscopy and by IR reflection-absorptio n spectroscopy (IRRAS) of monolayers in situ at the air/water (A/W) interfa ce. Bulk phase experiments revealed that AxV undergoes an irreversible ther mal denaturation at similar to 45-50 degrees C, as shown by the appearance of amide I bands at 1617 and 1682 cm(-1). However, some native secondary st ructure is retained, even at 60 degrees C, consistent with a partially unfo lded "molten globule" state. Formation of the Ca2+/phospholipid/protein ter nary complex significantly protects the protein from thermal denaturation a s compared to AxV alone, Ca2+/AxV, or lipid/AxV mixtures, Stabilization of AxV secondary structure by a DMPA monolayer in the presence of Ca2+ was als o observed by IRRAS, Spectra of an adsorbed AxV film in the presence or abs ence of Ca2+ showed a 10 cm(-1) shift in the amide I mode, corresponding to loss of ordered structure at the A/W interface. In both the bulk phase and IRRAS experiments, protection against H-D exchange in AxV was enhanced onl y in the ternary complex. The combined data suggest that the secondary stru cture of AxV is strongly affected by the Ca2+/membrane component of the ter nary complex whereas lipid conformational order is unchanged by protein.