Interaction of .alpha.-Lactalbumin with Phosphatidylglycerol. Influence of Protein Binding on the Lipid Phase Transition and Lipid Acyl Chain Mobility

Abstract

The mobility of spin-labeled lipids has been studied in dioleoyl and dimyristoyl phosphatidylglycerol bilayers and in their complexes with alpha-lactalbumin at pH 4.0, by using electron spin resonance (ESR) spectroscopy. The ESR spectra of phosphatidylglycerol spin-labeled at position 5 of the sn-2 chain indicate that association of alpha-lactalbumin with dimyristoyl phosphatidylglycerol bilayers increases the chain mobility at temperatures in the lipid gel phase, restricts the chain mobility at temperatures corresponding to the lipid fluid phase, and abolishes the cooperative lipid chain-melting transition. The ESR spectra of phosphatidylglycerols spin-labeled at eight different positions in the sn-2 chain show that binding of alpha-lactalbumin to dioleoyl phosphatidylglycerol bilayers at pH 4.0 causes a motional restriction throughout the full length of the lipid acyl chain. For phosphatidylglycerols spin-labeled at the terminal methyl end of the chains, a population of motionally restricted lipids that directly contacts membrane penetrant portions of the protein is detected. This population corresponds to 6.3 +/- 0.7 lipids/alpha-lactalbumin at saturation binding, and the high degree of motional restriction (maximum hyperfine splitting approximately 60 G) suggests that the protein may traverse the lipid bilayer. A small selectivity of phosphatidylglycerol over zwitterionic phospholipids for interaction with alpha-lactalbumin is found at subsaturating levels of binding at pH 4.0. Binding of alpha-lactalbumin also strongly restricts the motion of lipids spin-labeled in the polar head group region. These results are of direct relevance to the insertion and translocation of a protein in the molten globule state across lipid membranes.

Interaction of α-lactalbumin with lipids and possible implications for its emulsifying properties – A review

“…PC Fluorescence spectroscopy and circular dichroism Lala et al, 1995. DOPG and DMPG Electron spin resonance spectroscopy Montich & Marsh, 1995. a Abbreviations: DMPC, dimyristoylphosphatidylcholine; DMPG, dimyristoylphosphatidylglycerol; DOPG, 1,2-dioleylphosphatidylglycerol; DPPC, dipalmitoylphosphatidylcholine; DPPG, dipalmitoylphosphatidylglycerol; DSPS: 1,2-distearoyl-sn-glycero-3-[phospho-L-serine]; EPA, egg phosphatidic acid; EPC, egg phosphatidylcholine; EPS, egg phosphatidylserine; EYL, egg yolk lecithin; EYPC, egg yolk phosphatidylcholine; NBD-PE, N-(7-nitro-2,1,3-benzoxadiazol-4-yl) phosphatidylethanolamine; PBPS, porcine brain phosphatidylserine; PC, phosphatidylcholine; in solution, the protein was shown to be able to insert itself into the liquid condensed monolayer at an initial surface pressure of 39 AE 0.7 mN m À1 whereas in the presence of a Ca 2þ chelating agent, such as EDTA, the surface pressure was greater. These results show clearly the influence of the protein stability on its capacity of insertion into lipid monolayers.…”

“…The hydrophobic patches exposed on the a-LA molten globule surface are considered of key importance for the insertion of the protein into membranes (Chaudhuri et al, 2004;Lala et al, 1995;Montich & Marsh, 1995). In comparison with the apo form of the protein, it has been indicated that this latter inserts into lipid bilayer at slower kinetic than molten globule state (Chaudhuri et al, 2004).…”

“…Montich and Marsh (1995) studied the interaction of a-LA with spin-labelled lipid bilayers of dioleoyl and dimyristoyl phosphatydilglycerol using electron spin resonance (ESR) spectroscopy. They found that at pH 4, a-LA adopts a molten globule state and associates the lipid membranes affecting their chain mobility and packing.…”

“…It must be emphasized that the binding of the protein to lipid membranes increased their chain mobility at temperature corresponding to the lipid-gel transition (30 C), whereas at that of the lipid fluid phase (20 C), a limitation of the same mobility was observed. Moreover, the disruption of the cooperative chain packing of the membranes contributes to the abolition of the cooperative lipid chain-melting transition indicating therefore, that the protein may traverse the lipid bilayer (Montich & Marsh, 1995).…”

“…Effect of the pHAt pH values around 2, the adoption of the flexible molten globule state by bovine a-LA enables it to insert rapidly into bilayer mainly by hydrophobic interactions and by electrostatic bonds between the positive charges of the protein and the negative charged head groups of the phospholipids(Chaudhuri et al, 2004). Besides, hydrophobic interactions between the protein and the apolar lipids of the membrane could take place(Chaudhuri et al, 2004;Chenal et al, 2005;Herreman, Van Tornout, Van Cauwelaert, & Hanssens, 1981;Montich & Marsh, 1995).…”