Stability of annexin v in ternary complexes with ca2+ and anionic phospholipids:: IR studies of monolayer and bulk phases

Annexin V (AxV) is a member of a family of proteins that exhibit functionally relevant Ca2+-dependent binding to anionic phospholipid membranes. Protein structure and stability as a function of Ca2+ and phospholipids was studied by bulk phase infrared (IR) spectroscopy and by IR reflection-absorption spectroscopy (IRRAS) of monolayers in situ at the air/water (A/W) interface. Bulk phase experiments revealed that AxV undergoes an irreversible thermal 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 structure is retained, even at 60 degrees C, consistent with a partially unfolded "molten globule" state. Formation of the Ca2+/phospholipid/protein ternary complex significantly protects the protein from thermal denaturation as 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 also observed by IRRAS, Spectra of an adsorbed AxV film in the presence or absence 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 only in the ternary complex. The combined data suggest that the secondary structure of AxV is strongly affected by the Ca2+/membrane component of the ternary complex whereas lipid conformational order is unchanged by protein.