To characterize the interaction of peripheral proteins and membranes at the molecular level, we studied the reversible association of bovine ␣-lactalbumin (BLA) with lipid bilayers composed of different molecular forms of phosphatidylserine or equimolar mixtures of these phosphatidylserine forms and egg yolk phosphatidylcholine. At pH 4.5, almost all BLA (>90%) associates to negatively charged small unilamellar vesicles. The conformational changes that binding to these bilayers induced on the protein were characterized by circular dichroism and fluorescence spectroscopy. Because binding of BLA to negatively charged vesicles is reverted by adjusting the pH back to >6.0, we also investigated the conformation of the membrane-bound protein by NMRmonitored H-D exchange of the backbone amide protons. The conformation adopted by BLA bound to these bilayers resembles a molten globule-like state but the negative ellipticity at 222 nm and the apparent ␣-helix content of the bound protein senses the changes in the physical properties of the membrane. Binding to bilayers in the gel state appears to correlate with an increased amount of ␣-helical structure and with a lower extent of integration into the membrane, corresponding to the adsorbed protein, while the opposite is found for BLA bound to vesicles in the liquid-crystalline phase, corresponding to the embedded conformation. A common feature for the membrane-bound conformations of BLA is that the amphipathic helix C (residues 86 to 99) is an important determinant for the adsorption and further integration of the protein into the membrane.

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