Membrane-bound states of alpha-lactalbumin: implications for the protein stability and conformation.
alpha-Lactalbumin (alpha-LA) associates with dimyristoylphosphatidylcholine (DMPC) or egg lecithin (EPC) liposomes. Thermal denaturation of isolated DMPC or EPC alpha-LA complexes was dependent on the metal bound state of the protein. The intrinsic fluorescence of thermally denatured DMPC-alpha-LA was sensitive to two thermal transitions: the Tc 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 degrees 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-alpha-LA complex showed a distinct fluorescence thermal transition between 40 and 60 degrees 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 817:154-166). 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.
Cawthern
K M
KM
Department of Chemistry, Ohio State University, Columbus 43210, USA.
Permyakov
E
E
Berliner
L J
LJ
D000818:Animals; D002417:Cattle; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D007788:Lactose Synthase / Q000378:metabolism; D008081:Liposomes; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry
D008081:Liposomes; D008565:Membrane Proteins; D014364:Tryptophan; D007768:Lactalbumin; D007788:Lactose Synthase