Dehydration of the lipid-protein microinterface on binding of phospholipase A2 to lipid bilayers.
A novel method is described to demonstrate inaccessibility to the bulk aqueous phase of the microinterface between pig pancreatic phospholipase A2 and lipid bilayers to which this protein is bound. The method is based on the fact that the fluorescence emission quantum yields of the tryptophan residue of the protein and of a 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) chromophore attached to a lipid are lower in water as compared to that in deuterated water. The fluorescence emission quantum yield of these chromophores is measured in water and in deuterated water under conditions where the protein is either bound or not bound to the surface of a lipid bilayer containing the dansyl chromophore. Under conditions where the protein is tightly bound to the surface of the bilayer, desolvation of both fluorophores abolishes the observed effect of deuterated water. The tryptophan residue in the bound phospholipase A2 also becomes inaccessible to fluorescence quenching by acrylamide or succinimide. Desolvation of the microinterface is observed only under conditions that are significant for the catalytic action of phospholipase A2 in the scooting mode and not in the hopping mode. Also, under similar conditions, binding of pro-phospholipase A2 to anionic vesicles does not cause dehydration of the microinterface. The mechanistic significance of these observations for lipid-protein interactions, in general, and for interfacial catalysis and interfacial activation, in particular, is discussed.
Jain
M K
MK
Department of Chemistry, University of Delaware, Newark 19716.
Vaz
W L
WL
D000818:Animals; D005453:Fluorescence; D008051:Lipid Bilayers / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding; D013552:Swine; D014364:Tryptophan
D008051:Lipid Bilayers; D014364:Tryptophan; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2