Stability of Annexin V in Ternary Complexes with Ca super(2+) and Anionic Phospholipids: IR Studies of Monolayer and Bulk Phases

• Published in: Biochemistry (Easton) Vol. 38; no. 2; pp. 792 - 799
• Main Authors: Wu, Fangjun, Flach, C R, Seaton, BA, Mealy, T R, Mendelsohn, R
• Format: Journal Article
• Language: English
• Published: 01.01.1999
• ISSN: 0006-2960

Annexin V (AxV) is a member of a family of proteins that exhibit functionally relevant Ca super(2+)-dependent binding to anionic phospholipid membranes. Protein structure and stability as a function of Ca super(2+) 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 degree C, as shown by the appearance of amide I bands at 1617 and 1682 cm super(-1). However, some native secondary structure is retained, even at 60 degree C, consistent with a partially unfolded "molten globule" state. Formation of the Ca super(2+)/phospholipid/protein ternary complex significantly protects the protein from thermal denaturation as compared to AxV alone, Ca super(2+)/AxV, or lipid/AxV mixtures. Stabilization of AxV secondary structure by a DMPA monolayer in the presence of Ca super(2+) was also observed by IRRAS. Spectra of an adsorbed AxV film in the presence or absence of Ca super(2+) showed a 10 cm super(-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 arrow right 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 Ca super(2+)/membrane component of the ternary complex whereas lipid conformational order is unchanged by protein.