Interaction of Alzheimer beta-amyloid peptide(1-40) with lipid membranes

The beta-amyloid peptide beta AP(1-40), a 40-amino acid residues peptide, is one of the major components of Alzheimer's amyloid deposits. beta AP(1-40) exhibits only a limited solubility in aqueous solution and undergoes a concentration-dependent, cooperative random coil reversible beta-structure transition for Cpep < 10 microM [Terzi, E., Holzemann, G., and Seelig, J. (1995) J. Mol. Biol. 252, 633-642]. In the presence of acidic lipid, the equilibrium is shifted further toward beta-structured aggregates. We have now characterized the lipid-peptide interaction using circular dichroism (CD) spectroscopy, lipid monolayers, and deuterium and phosphorus-31 solid-state nuclear magnetic resonance (NMR). CD spectroscopy revealed a distinct interaction between beta AP(1-40) and negatively charged unilamellar vesicles. In addition to the random coil reversible beta-structured aggregate equilibrium at low lipid-to-peptide (L/P) ratios, a beta-structure --<alpha-helix transition was observed at L/P < 55. beta AP(1-40) was found to insert into acidic monolayers provided the lateral pressure was low (20 mN/m). The extent of incorporation increased distinctly with the content of acidic lipid in the monolayer. However, at a lipid packing density equivalent to that of a bilayer (lateral pressure < or = 32 mN/m), no insertion of beta AP(1-40) was observed. The lipid molecular structure in the presence of beta AP(1-40) was studied with NMR. Phosphatidylcholine (PC) was selectively deuterated at the choline headgroup and at the cis-double bond of the oleic acyl chain and mixed with phosphatidylglycerol (PG). Phosphorus-31 NMR showed that the lipid phase retained the bilayer structure at all lipid-to-protein ratios. Deuterium NMR revealed no change in the headgroup conformation of the choline moiety or in the flexibility and ordering of the hydrocarbon chains upon the addition of beta AP-(1-40). It can be concluded that beta AP(1-40) binds electrostatically to the outer envelope of the polar headgroup region without penetrating between the polar groups. The data suggest a new mechanism of helix formation induced by the proper alignment of five positive charges of beta AP(1-40) on the negatively charged membrane template

Self-association of beta-amyloid peptide (1-40) in solution and binding to lipid membranes

The beta-amyloid peptide (beta AP), a 39 to 43 residue peptide, is the major component of Alzheimer plaques. Using circular dichroism spectroscopy, titration calorimetry, and analytical ultracentrifugation we have analyzed the self-association of beta AP(1-40) in aqueous solution and the binding of beta AP(1-40) to negatively charged lipid vesicles. The CD spectra of both aggregation and membrane binding are characterized by an isodichroic point at 212 nm, indicating a simple two-state equilibrium for both cases. In aqueous solution beta AP(1-40) exhibits a reversible, concentration-dependent random coilbeta-structure transition which can be described by a cooperative aggregation model with an association constant of s = 1.05 x 10(4)M-1 and a nucleation parameter of sigma = 0.012. A similar conformational change is observed upon addition of lipid. At a given peptide concentration, the addition of negatively charged, small unilamellar vesicles also induces a conformational change from a random coil conformation to a conformation with 40 to 60% beta-structure. The binding isotherm can be measured with high sensitivity titration calorimetry. It is approximately linear in the initial binding phase and exhibits an apparent saturation behaviour. The apparent binding constant decreases with concentration from Kapp approximately 2100 M-1 at low concentration to 700 M-1 at the highest concentration measured. Peptide penetration into the lipid membrane and peptide aggregation at the membrane surface are proposed as possible mechanisms to explain the lipid-induced random coilbeta-structure transition

Alzheimer beta-amyloid peptide 25-35 : electrostatic interactions with phospholipid membranes

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 beta AP(25-35)OH and beta AP(25-35Nle)NH2 exhibit a reversible random-coilbeta-sheet (or beta-structured aggregate) transition. Addition of lipid vesicles containing negatively charged lipids shifts the random-coilbeta-sheet equilibrium almost completely toward beta-sheet structure, which can be explained by the specific conditions created at 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 beta-sheet formation. The third peptide, beta AP-(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 delta H approximately -2 kcal/mol for beta AP(25-35)OH and -8 kcal/mol for beta AP(25-35)NH2. The apparent binding constants, calculated with bulk concentrations, were large and varied between 500 and 5 x 10(4) M-1, 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 approximately 2-10 M-1.(ABSTRACT TRUNCATED AT 250 WORDS

Reversible random coil-beta-sheet transition of the Alzheimer beta-amyloid fragment (25-35)

The beta-amyloid protein (39-43 amino acid residues) is the major constituent of the amyloid deposits found in brain of patients with Alzheimer's disease. Using circular dichroism spectroscopy, we have studied the secondary structure and the aggregation of fragment 25-35 of the beta-amyloid protein (beta AP(25-35)OH) under a variety of conditions. beta AP(25-35)OH in solution at pH 4.0 or 5.5 exhibits a concentration-dependent random coilbeta-sheet transition. The equilibrium is characterized spectroscopically by an isodichroic point and can be described quantitatively by a simple association model with association constants between 1.8 x 10(4) M-1 (non-cooperative model, nucleation parameter sigma = 1) and 2.9 x 10(4) M-1 (cooperative model, sigma = 0.2). The enthalpy of association is delta H approximately -3 kcal/mol as determined by titration calorimetry. The equilibrium is shifted completely toward beta-structured fibrils at pH 7.4 where the Met-35 carboxyl group is fully charged. In contrast, removal of the charged carboxy terminus by amidation locks the equilibrium in the random coil conformation. Model calculations suggest an antiparallel beta-sheet structure involving residues 28-35 which is stabilized at both ends of the beta-sheet by ion pairs formed between Lys-28 and Met-35. Removal of fibrils via millipore filtration leads to solutions with random coil monomers only. Seeding these solutions with a few fibrils establishes a new random coilbeta-sheet equilibrium

Binding of substance P agonists to lipid membranes and to the neurokinin-1 receptor

Three new analogues of the neuropeptide substance P (SP) were synthesized. The C-terminal message segment was made more hydrophilic in (Arg9)SP or more hydrophobic in (Nle9)SP. In (AcPro2, Arg9)SP the charge at the N-terminal address segment was reduced, while that of the message segment was increased. The rationale underlying these substitutions was to correlate the physical-chemical properties of the SP-analogues, in particular their lipid-induced conformation and membrane-binding affinity, with receptor binding and functional activity. In solution, all three analogues exhibited random coil conformations as evidenced by circular dichroism spectroscopy. Addition of SDS micelles induced partially alpha-helical structures. The same structure was also produced by negatively charged lipid vesicles for (AcPro2, Arg9)SP and (Arg9)SP whereas both alpha-helix-like structures and beta-sheet structures were observed for SP and (Nle9)SP. The measurement of the Gibbs adsorption isotherms and monolayer expansion studies provided quantitative data on the surface area requirement and on the membrane penetration area of the SP analogues. The thermodynamic parameters for lipid binding were determined with monolayer expansion for measurements and high-sensitivity titration calorimetry. The apparent binding constants, Kapp, for membranes containing 100% POPG were of the order of 10(3)- 10(5) M(-1). The binding was due to electrostatic attraction of the cationic peptides to the negatively charged membrane surface. The intrinsic (hydrophobic) binding constants, obtained after correcting for electrostatic effects, were much smaller with Kp=10+/- 1 M(-1) for (Arg9)SP, 9 +/- 1 M(-1) for (AcPro2, Arg9)SP, and 39 +/- 3 M(-1) for (Nle9)SP. The measurement of the binding affinities to the NK-1 receptor and of the in vitro activities showed that all three peptides behaved as agonists. Their binding affinity to the neurokinin-1 receptor decreased with the size of the side chains at position 9 of the amino acid sequence but was independent of the cationic charge of the peptides. The fact that even the highly charged (Arg9)SP has agonistic activity provides evidence that the binding epitope at the receptor is in a rather hydrophilic environment. This finding is in agreement with the low hydrophobic binding constants and the weak penetration of the three peptides into negatively charged membranes. It argues against a membrane mediated receptor mechanism and suggests that the agonist approaches the receptor binding, site from the aqueous phase