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The interaction between proteins and membranes is of great interest in biomedical and biotechnological research for its implication in many functional and dysfunctional processes. We present an experimental study on the interaction between model membranes and alpha-lactalbumin (alpha-La). alpha-La is widely studied for both its biological function and its anti-tumoral properties. We use advanced fluorescence microscopy and spectroscopy techniques to characterize alpha-La-membrane mechanisms of interaction and alpha-La-induced modifications of membranes when insertion of partially disordered regions of protein chains in the lipid bilayer is favored. Moreover, using fluorescence lifetime imaging, we are able to distinguish between protein adsorption and insertion in the membranes. Our results indicate that, upon addition of alpha-La to giant vesicles samples, protein is inserted into the lipid bilayer with rates that are concentration-dependent. The formation of heterogeneous hybrid protein-lipid co-aggregates, paralleled with protein conformational and structural changes, alters the membrane structure and morphology, leading to an increase in membrane fluidity.

International audience; Sperm whale myoglobin can be considered as the model protein of the globin family. The pH-dependence of the interactions of apomyoglobin with lipid bilayers shares some similarities with the behavior of pore-forming domains of bacterial toxins belonging also to the globin family. Two different states of apomyoglobin bound to a lipid bilayer have been characterized by using hydrogen/deuterium exchange experiments and mass spectrometry. When bound to the membrane at pH 5.5, apomyoglobin remains mostly native-like and interacts through alpha-helix A. At pH 4, the binding is related to the stabilization of a partially folded state. In that case, alpha-helices A and G are involved in the interaction. At this pH, alpha-helix G, which is the most hydrophobic region of apomyoglobin, is available for interaction with the lipid bilayer because of the loss of the tertiary structure. Our results show the feasibility of such experiments and their potential for the characterization of various membrane-bound states of amphitropic proteins such as pore-forming domains of bacterial toxins. This is not possible with other high-resolution methods, because these proteins are usually in partially folded states when interacting with membranes.

International audience; 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.