Membrane‐bound states of α‐lactalbumin: Implications for the protein stability and conformation

Abstract

Abstracta-Lactalbumin (a-LA) associates with dimyristoylphosphatidylcholine (DMPC) or egg lecithin (EPC) liposomes. Thermal denaturation of isolated DMPC or EPC a-LA complexes was dependent on the metal bound state of the protein. The intrinsic fluorescence of thermally denatured DMPC-a-LA was sensitive to two thermal transitions: the T,. of the lipid vesicles, and the denaturation of the protein. Quenching experiments suggested that tryptophan accessibility increased upon protein-DMPC association, in contrast with earlier suggestions that the limited emission red shift upon association with the liposome was due to partial insertion of tryptophan into the apolar phase of the bilayer (Hanssens I et al., 1985, Biochim Biophys Acta 817:154-166). On the other hand, above the protein transition (70 "C), the spectral blue shifts and reduced accessibility to quencher suggested that tryptophan interacts significantly with the apolar phase of either DMPC and EPC. At pH 2, where the protein inserts into the bilayer rapidly, the isolated DMPC-a-LA complex showed a distinct fluorescence thermal transition between 40 and 60 "C, consistent with a partially inserted form that possesses some degree of tertiary structure and unfolds cooperatively. This result is significant in light of earlier findings of increased helicity for the acid form, i.e., molten globule state of the protein (Hanssens I et al., 1985, Biochim Biophys Acta 812154-166). These results suggest a model where a limited expansion of conformation occurs upon association with the membrane at neutral pH and physiological temperatures, with a concomitant increase in the exposure of tryptophan to external quenchers; i.e., the current data do not support a model where an apolar, tryptophan-containing surface is covered by the lipid phase of the bilayer.

Interactions of α-Lactalbumin with Fatty Acids and Spin Label Analogs

“…The intrinsic fluorescence emission red shift and general increase resembles studies in which apo-␣-LA was transferred from aqueous media and embedded in the surface of liposomes (11,12). Although there are several mechanisms to explain the fluorescence changes, one model could even be consistent with identical binding sites for both stearic acid and 5-DSA.…”

Susceptibility of lysozyme to in-vitro digestion and immunoreactivity of its digests

“…Hen egg white LYS and bovine LA are homologous to each other as they share similar primary structures, but their unfolding profiles and the stabilities of their native conformations are very different (Cawthern, Permyakov, & Berliner, 1996;Polverino de Laureto et al, 2002). The biological functions of LYS, such as its antimicrobial and immunomodulating properties, have been attributed to its ability to interact with membrane component phospholipids and to penetrate into lipid bilayers, aspects which have been broadly studied (Gorbenko, Loffe, & Kinnunen, 2007;Yuan et al, 2007).…”

Phospholipid-Cytochrome c Interaction

“…These authors concluded that specific hydrophobic interactions with the sn-2 acyl chain are a major determinant for the binding of annexin V to membranes (35). Finally, the lipid binding properties of milk protein ␣-lactalbumin closely resemble those of cyt c (36). Moreover, lipid binding to ␣-lactalbumin has been suggested to induce the molten globule state, similarly to cyt c (37,38).…”