Annexin V binding to the outer leaflet of small unilamellar vesicles leads to altered inner-leaflet properties: 31P- and 1H-NMR studies.
Calcium-dependent binding to phospholipid membranes is closely associated with annexin functional properties. In these studies, 31P- and 1H-nuclear magnetic resonance (NMR) experiments have been performed to study the effects of binding of recombinant rat annexin V to sonicated small unilamellar vesicles (SUVs). High-resolution 31P-NMR spectra of SUVs containing mixtures of synthetic phosphatidic acid (PA) and phosphatidylcholine (PC) show resolvable resonances corresponding to the inner-leaflet PA, outer-leaflet PA, and PC phosphoryl groups. When annexin binding occurs, the outer-leaflet PA 31P resonance shifts while that of PC is unaffected, consistent with selective binding of the protein to the phosphoryl moiety of the PA component. Further, annexin V binding to membrane outer-leaflet phospholipids has a measurable effect on inner-leaflet phospholipids of intact vesicles. 1H-NMR T1 relaxation measurements of SUVs containing acyl-chain-perdeuterated PC show no effects on the PA hydrocarbon-chain segmental motions upon annexin binding. Circular dichroism measurements indicate that the protein does not undergo a significant conformational change upon binding to the vesicles. The observed NMR changes do not correspond to proton or calcium gradients, nor to lateral segregation of extended patches of homogeneous phospholipids. The combined evidence suggests that selective, peripheral annexin-membrane interactions influence the environment of the inner vesicular surface. The mechanism proposed is a protein-induced change in vesicle morphology that corresponds to reduced curvature.
Swairjo
M A
MA
Department of Physiology, Boston University School of Medicine, Massachusetts 02118.
Roberts
M F
MF
Campos
M B
MB
Dedman
J R
JR
Seaton
B A
BA
D000818:Animals; D017304:Annexin A5 / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004295:Dihydroxyphenylalanine; D004134:Dimyristoylphosphatidylcholine; D004926:Escherichia coli / Q000235:genetics; D008051:Lipid Bilayers; D009682:Magnetic Resonance Spectroscopy; D010759:Phosphorus Isotopes; D011485:Protein Binding; D011522:Protons; D051381:Rats
D017304:Annexin A5; D008051:Lipid Bilayers; D010759:Phosphorus Isotopes; D011522:Protons; D004295:Dihydroxyphenylalanine; D004134:Dimyristoylphosphatidylcholine