Differential Interaction of Bovine Alpha-Lactalbumin with Membranes: Interplay of Negatively Charged Lipids and Cholesterol

Abstract

Many soluble proteins are known to interact with membranes and the mechanism of such interactions in cellular processes is beginning to be understood. Bovine alpha-lactalbumin (BLA) is a soluble protein and yet it interacts with membranes. We recently reported the specific binding of apo-BLA with negatively charged membranes using a variety of fluorescence approaches [1]. A novel finding is that BLA exhibits an enhanced binding to negatively charged membranes in the presence of cholesterol and it possesses cholesterol recognition/interaction amino acid consensus (CRAC), a motif recognized for preferential association with cholesterol in many proteins. We monitored the specificity of sterol interaction by replacing cholesterol with 7-dehydrocholesterol (an immediate biosynthetic precursor of cholesterol), and observed that BLA-sterol interaction is specific to membrane cholesterol. Significant Fluorescence Resonance Energy Transfer (FRET) was observed between the tryptophan residues of BLA and dehydroergosterol (a naturally occurring fluorescent analog of cholesterol) in presence of negatively charged membranes, indicating close proximity between them. Dipole potential measurements upon BLA binding to membranes and docking studies of cholesterol with BLA provide useful insight into the lipid selectivity of BLA binding to membranes. Depth analysis by the parallax approach provides evidence for interfacial localization of tryptophans of BLA when bound to membranes. Taken together, our results assume significance in the light of tumoricidal and antimicrobial functions of α-lactalbumin [2,3].