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 approximately 45-50 degreesC, 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 degreesC, 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.
Wu
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F
Department of Chemistry, Newark College, Rutgers University, New Jersey 07102, USA.
Flach
C R
CR
Seaton
B A
BA
Mealy
T R
TR
Mendelsohn
R
R
D000818:Animals; D000838:Anions / Q000737:chemistry; D017304:Annexin A5 / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry*; D046911:Macromolecular Substances; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry*; D017433:Protein Structure, Secondary; D051381:Rats; D013055:Spectrophotometry, Infrared
D000838:Anions; D017304:Annexin A5; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010715:Phosphatidylglycerols; D010743:Phospholipids; C057561:1-palmitoyl-2-oleoylphosphatidylethanolamine; C002773:dimyristoylphosphatidylglycerol; C017251:1,2-oleoylphosphatidylcholine; D002118:Calcium