Stability of annexin V in ternary complexes with CA2+ and anionic phospholipids: IR studies of monolayer and bulk phases

Fangjun Wu, Carol R. Flach, Barbara A. Seaton, Tanya R. Mealy, Richard Mendelsohn

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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 ~45-50 °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 °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.

Original languageEnglish (US)
Pages (from-to)792-799
Number of pages8
Issue number2
StatePublished - Jan 12 1999

All Science Journal Classification (ASJC) codes

  • Biochemistry


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