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Interactions of apomyoglobin with membranes: Mechanisms and effects on heme uptake

Laboratoire de Biophysique Moléculaire et Cellulaire, Unité Mixte de Recherche 5090, Département Réponse et Dynamique Cellulaires, CEA-Grenoble, 38054 Grenoble cedex 9, France

(RECEIVED August 30, 2006; FINAL REVISION November 22, 2006; ACCEPTED November 27, 2006)

The last step of the folding reaction of myoglobin is the incorporation of a prosthetic group. In cells, myoglobin is soluble, while heme resides in the mitochondrial membrane. We report here an exhaustive study of the interactions of apomyoglobin with lipid vesicles. We show that apomyoglobin interacts with large unilamellar vesicles under acidic conditions, and that this requires the presence of negatively charged phospholipids. The pH dependence of apomyoglobin interactions with membranes is a two-step process, and involves a partially folded state stabilized at acidic pH. An evident role for the interaction of apomyoglobin with lipid bilayers would be to facilitate the uptake of heme from the outer mitochondrial membrane. However, heme binding to apomyoglobin is observed at neutral pH when the protein remains in solution, and slows down as the pH becomes more favorable to membrane interactions. The effective incorporation of soluble heme into apomyoglobin at neutral pH suggests that the interaction of apomyoglobin with membranes is not necessary for the heme uptake from the lipid bilayer. In vivo, however, the ability of apomyoglobin to interact with membrane may facilitate its localization in the vicinity of the mitochondrial membranes, and so may increase the yield of heme uptake. Moreover, the behavior of apomyoglobin in the presence of membranes shows striking similarities with that of other proteins with a globin fold. This suggests that the globin fold is well adapted for soluble proteins whose functions require interactions with membranes.

Keywords: apomyoglobin; protein/membrane interactions; large unilamellar vesicles; amphitropic proteins; globin fold; heme uptake; protein folding

² Present addresses: Harvard Medical School, Department of Microbiology and Molecular Genetics, 200 Longwood Avenue, Boston, MA 02115, USA.

³ Biochemistry of Macromolecular Interactions Unit, Pasteur Institute, 75724 Paris Cedex 15, France.

Reprint requests to: Vincent Forge, Laboratoire de Biophysique Moléculaire et Cellulaire, Unité Mixte de Recherche 5090, Département Réponse et Dynamique Cellulaires, CEA-Grenoble, 17 Rue des Martyrs, 38054 Grenoble cedex 9, France; e-mail: vincent.forge{at}cea.fr; fax: 04-38-78-54-87.

Abbreviations: aMb: sperm whale apomyoglobin; dT: diphtheria toxin translocation domain; LUVs: large unilamellar vesicles; ^–LUVs: anionic LUVs; ^±LUVs: zwitterionic LUVs; EPC: egg phosphatidylcholine; EPA: egg phosphatidic acid; Trp: tryptophan.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062531207.

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