MEMBRANE-BOUND STATES OF ALPHA-LACTALBUMIN - IMPLICATIONS FOR PROTEINSTABILITY AND CONFORMATION

alpha-Lactalbumin (alpha-LA) associates with dimyristoylphosphatidylch oline (DMFC) or egg lecithin (EPC) liposomes. Thermal denaturation of isolated DMPC or EPC alpha-LA complexes was dependent on the metal bou nd state of the protein. The intrinsic fluorescence of thermally denat ured DMPC-alpha-LA was sensitive to two thermal transitions: the T-c o f the lipid vesicles, and the denaturation of the protein. Quenching e xperiments suggested that tryptophan accessibility increased upon prot ein-DMPC association, in contrast with earlier suggestions that the li mited 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 o ther hand, above the protein transition (70 degrees C), the spectral b lue shifts and reduced accessibility to quencher suggested that trypto phan 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-alpha-LA complex showed a distinct fluorescence thermal transition between 40 and 60 degrees C, consistent with a partially in serted form that possesses some degree of tertiary structure and unfol ds cooperatively. This result is significant in light of earlier findi ngs of increased helicity for the acid form, i.e., molten globule stat e of the protein (Hanssens I et al., 1985, Biochim Biophys Acta 817: 1 54-166). These results suggest a model where a limited expansion of co nformation occurs upon association with the membrane at neutral pH and physiological temperatures, with a concomitant increase in the exposu re of tryptophan to external quenchers; i.e,, the current data do not support a model where an apolar, tryptophan-containing surface is cove red by the lipid phase of the bilayer.