Annexins are soluble proteins that are best known for their ability to undergo reversible Ca(2+)-dependent binding to the surface of phospholipid bilayers. Recent studies, however, have shown that annexins also reversibly bind to membranes in a Ca(2+)-independent manner at mildly acidic pH. We investigated the structural changes that occur upon pH-dependent membrane binding by performing a nitroxide scan on the helical hairpin encompassing helices A and B in the fourth repeat of annexin B12. Residues 251-273 of annexin B12 were replaced, one at a time, with cysteine and then labeled with a nitroxide spin label. Electron paramagnetic resonance (EPR) mobility and accessibility analyses of soluble annexin B12 derivatives were in excellent agreement with the known crystal structure of annexin B12. However, EPR studies of annexin B12 derivatives bound to membranes at pH 4.0 indicated major structural changes in the scanned region. The helix-loop-helix structure present in the soluble protein was converted into a continuous transmembrane alpha-helix that was exposed to the hydrophobic core of the bilayer on one side and exposed to an aqueous pore on the other side. Asp-264 was on the hydrophobic membrane-exposed face of the amphipathic transmembrane helix, thereby suggesting that protonation of its carboxylate group stabilized the transmembrane form. Inspection of the amino acid sequence of annexin B12 revealed several other helical hairpin regions that might refold and form continuous amphipathic transmembrane helices in response to protonation of Asp or Glu switch residues on or near the hydrophobic face of the helix.