Membrane Interaction of N-Terminal Peptides of Annexin A1

Abstract

Annexin A1 is a member of a family of calcium-dependent membrane-binding proteins. This protein is known for its anti-inflammatory effect and for its ability to aggregate phospholipid membranes. A N-terminal annexin A1 peptide activates and desensitizes the human N-formyl peptide receptor (FPR), a member of G-protein coupled receptors that is a key modulator of chemotaxis directing granulocytes toward sites of bacterial infections. The N-terminal domain of annexin A1 mainly seems to be responsible for the biological activities of this protein. So we are interested in the (Ca2+-independent) membrane binding activities and structural information of the phospholipid membrane interaction of N-terminal peptides of annexin A1. We investigated the membrane interaction of synthesized N-terminal peptides of annexin A1 (residues Ac2-26 and 1-40) with artificial vesicles of different lipid compositions (mainly phosphatidylserine and phosphatidylcholine) and under varying buffer conditions (ionic strength, pH of the solution) by using fluorescence techniques. For these measurements we used the A1NT peptide labelled at 4 different positions with a dansyl fluorophore. From the experimentally measured binding curves the Gibbs free energy for the peptide transfer from aqueous solution to the lipid membrane was calculated. The effective charge of the peptide depends on the pH value of the buffer and is about half of its theoretical net charge. Fluorescence correlation spectroscopy measurements were done with TAMRA labelled A1NT peptide and giant unilamellar vesicles. The binding of fluorescently labelled peptides to micro-domains (lipid rafts) in differently compounded giant vesicles we observed using confocal laser scanning microscopy. Structural information of membrane bound peptide we reached by polarized infrared spectroscopy (ATR-FTIR). Further, the position of A1NT α-helix in the membrane was estimated from the intrinsic tyrosine fluorescence, from quenching experiments with spin labelled phospholipids using A1NT Trp.