Structural requirements for the association of native and partially folded conformations of alpha-lactalbumin with model membranes

The effect of the structure and stability of several conformers of alpha-lactalbumin in aqueous solution on their association to negatively charged large unilamellar vesicles has been studied by circular dichroism, infrared spectroscopy, differential scanning calorimetry, and by content leakage experiments. Our results indicate that the affinity of alpha LA for negatively charged vesicles strongly depends on its conformational properties in solution. Analysis of the pH dependence of the interaction for the different conformers reveals that native-like, calcium-bound, ordered conformations become bilayer-associated through electrostatic fords. However, partially folded conformers are able to interact with negatively charged membranes at pHs higher than the protein isoelectric point, suggesting that hydrophobic interactions brought about by the exposure of hydrophobic residues at the protein surface are able to overcome the unfavorable electrostatic repulsion, Calorimetric and spectroscopic data in solution also indicate that substantial protein destabilization facilitates its subsequent membrane binding, and that the association process is favored for a set of conformers having significant secondary structure, but lacking native-like, stable tertiary structure. Aggregation of the unfolded cl-lactalbumin molecules and burial of hydrophobic surfaces: upon formation of ordered tertiary structure significantly reduce their membrane perturbing activity. These observations suggest that formation of a flexible structural intermediate of alpha-lactalbumin in solution is a prerequisite for Its association with membranes.