Alzheimer , & Amyloid Peptide 25-35 : Electrostatic Interactions with Phospholipid Membranest

The role of lipids in the aggregation of three Alzheimer model peptides was investigated with circular dichroism spectroscopy and high-sensitivity titration calorimetry under conditions of low ionic strength. In solution, the peptides PAP(25-35)OH and @AP(25-35Nle)NH2 exhibit a reversible randomcoil F! @-sheet (or @-structured aggregate) transition. Addition of lipid vesicles containing negatively charged lipids shifts the random-coil + @-sheet equilibrium almost completely toward @-sheet structure, which can be explained by the specific conditions created a t the membrane surface: the cationic peptides are attracted to the negatively charged membrane, and the increase in peptide concentration together with the partial alignment of the peptide molecules then facilitates @-sheet formation. The third peptide, PAP(25-35)NH2, also binds to the lipid membrane but was found to adopt an essentially random-coil structure, both with and without lipids. A quantitative characterization of the binding equilibrium was possible with high-sensitivity titration calorimetry. All three peptides exhibited exothermic binding enthalpies which varied between Lw= -2 kcal/mol for PAP(25-35)OH and-8 kcal/mol for BAP(25-35)NHz. The apparent binding constants, calculated with bulk concentrations, were large and varied between 500 and 5 X lo4 M-l, depending on the experimental conditions. However, after correction for electrostatic charge effects using the Gouy-Chapman theory, the intrinsic binding constants were found to be constant and much smaller with K 2-10 M-l. The low intrinsic binding constants exclude significant hydrophobic interactions between the lipid membrane and the three Alzheimer model peptides even though residues 29-35 are considered to be part of a membrane-spanning domain in the full-length precursor protein. The predominance of electrostatic forces was demonstrated by addition of 0.1 M NaCl, which abolished the peptide-lipid interaction. Alzheimer's disease, the degenerative disorder of the human brain, is characterized pathologically by abnormal depositions of protein in the brain. The major protein component is the @-amyloid peptide (BAP),' an amphiphilic peptide consisting of 39-43 amino acids. @AP is a proteolytic fragment of the membrane-bound @-amyloid precursor protein (@APP) which is cleaved by an as yet unidentified protease (Kang et al., 1987). In OAPP, residues 1-28 of PAP constitute an extracellular domain whereas residues 29-39(43) form a transmembrane domain [cf. Fraser et al. (1 99 1) and Selkoe (1993)l. No quantitative data exist on the binding of @AP itself to lipid membranes. Recently, PAP has been incorporated into phosphatidylserine liposomes, inducing the formation of Ca2+-dependent channels when fused with planar lipid bilayers (Arispe et al., 1993). The role of the putative transmembrane domain is, however, unclear. @AP fibrils are characterized by a high percentage of 0-sheet structure whereas monomeric @AP adopts a predominantly random-coil conformation in solution [cf. Lansbury (1992), Barrow and Zagorski (1991), and Barrow et al. (1992)l. "Membrane-mimicking" solvents such as trifluoroethanol 7 Supported by a FEBS long-term fellowship (E.T.) and by Swiss t Biocenter of the University of Basel. 8 E. Merck. @ Abstract published in Advance ACS Abstracts, May 15, 1994. Abbreviations: POPC, l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; A&Na, sodium acetate; MOPS, morpholinopropanesulfonic acid; TRIS, tris(hydroxymethy1)aminomethane; CD, circular dichroism; @AP, @-amyloid peptide; OAPP, @-amyloid precursor protein; SP, substance P SUV, small unilamellar vesicle. National Science Foundation Grant 31.27505. 89. 0006-2960/94/0433-1434%04.50/0 (TFE) have been found to induce either an a-helical structure or an oligomeric @-structure of PAP and also of some of its fragments (Barrow & Zagorski, 1991; Otvos et al., 1993), suggesting that similar conformational changes could be triggered upon interaction of PAP with lipid membranes. The relevance of these conformational transitions for membrane systems has, however, not been demonstrated experimentally. We have previously shown that peptide PAP(25-35)OH in solution undergoes a concentration-dependent random-coil * @-sheet transition at pH 4.0 and 5.5 (Terzi et al., 1994). At pH 7.4, the equilibrium is shifted completely toward the @-sheet conformation whereas removal of the negative charge by amidation of the carboxy terminal stabilizes the randomcoil conformation. A quantitative description of the randomcoil @-sheet transition was possible in terms of a simple cooperative association model. We have now investigated the influence of neutral and negatively charged lipid vesicles on the conformational equilibrium of PAP(25-35)OH and two related derivatives with CD spectroscopy and titration calorimetry. The selection of fragment PAP(25-35) for membrane binding studies is attractive for two reasons. On the one hand, @AP(25-35) comprises six amino acids of the putative transmembrane region which could be relevant for membrane binding. On the other hand, the C-terminal sequence of PAP(25-35)"~ (X-Gly-Leu-Met-"2) is identical to that of substance P and other tachykinin neuropeptides (Yankner et al., 1990). Since the binding of substance P to lipid membranes has been investigated in detail with various physical-chemical techniques (Seelig & Macdonald, 1989; Seelig, 1992), this facilitates quantitative comparison between the PAP fragments and substance P. The