Structural Evolution and Membrane Interaction of the 40-Residue β Amyloid Peptides: Differences in the Initial Proximity between Peptides and the Membrane Bilayer Studied by Solid-State Nuclear Magnetic Resonance Spectroscopy

Abstract

Interactions between the β amyloid (Aβ) peptides and cellular membranes have severe consequences such as neuronal cell disruption and therefore may play important roles in Alzheimer's disease. Understanding the structural basis behind such interactions, however, is hindered by the complexity of the Aβ-membrane systems. In particular, because the Aβ peptides are partially incorporated in the membrane bilayer after enzymatic cleavage, there are multiple possibilities in terms of the initial proximity between the peptides and membranes. Structural studies using in vitro model systems with either externally added or preincorporated Aβ in membrane bilayers resulted in distinct evolution pathways. Previous work has shown that the externally added Aβ formed long and mature filaments, while preincorporated Aβ generated short and curvy fibrils. In this study, we perform detailed characterizations on the structural evolution and membrane interaction for these two pathways, using a combination of solid-state nuclear magnetic resonance spectroscopy and other techniques. For the externally added Aβ, we determined the residue-specific structural evolution during the fibrillation process. While the entire fibrillation process for the externally added Aβ was slow, the preincorporated Aβ generated Aβ-lipid complexes rapidly. Specific interactions between the lipids and peptides were observed, suggesting the colocalization of lipids and peptides within the complex. Formation of such a complex induced molecular-level changes in the lipid bilayer, which may serve as a possible mechanism of membrane disruption.

Distinct Membrane Disruption Pathways Are Induced by 40-Residue β-Amyloid Peptides

“…This result is surpris- ing because we have proposed previously (and will prove with later evidence) that membranes are seriously disrupted by the elution of A␤ oligomers under this condition (30,31). However, it is worth noting that the calcein-contained liposomes used in this fluorescence assay were separated from the uncaptured calcein through slow dialysis (see "Experimental Procedures").…”

“…2B). Therefore, high-order A␤ oligomers, but not fibrils (proved by transmission electron microscopy (TEM) in previous studies (31)), seem to be present as the dominant form of A␤ in the preincorporation samples. These oligomers may form during liposome preparation when a high concentration of A␤ peptides is suspended in aqueous buffer.…”

“…A␤-membrane Sample Preparation-"External addition" and "preincorporation" sample preparation protocols were described in detail previously (23,30,31). Briefly, the external addition samples were made by adding A␤ to preformed liposomes, which were prepared by resuspending dried lipid film in phosphate buffer (10 mM (pH 7.4) with 0.01% NaN 3 ), followed by 10 cycles of freeze-thaw and 30ϫ extrusion with the designed membrane sizes.…”

“…For magic angle spinning spectroscopy, the spinning frequency was set to 10 kHz, and the temperature for all NMR measurements was controlled with a 270 K cooling N 2 flow (corresponding to the ϳ280 K sample temperature estimated from the 1 H chemical shift of H 2 O). The REDOR curves were fit to a model 13 C, 31 P two-spin system using a SIMPSON simulation package and the best fit internuclear distances were reported (39).…”

Phosphorylation Interferes with Maturation of Amyloid-β Fibrillar Structure in the N Terminus

“…Local perturbation of A␤ conformation by Ser 8 phosphorylation, as demonstrated by our data, can therefore be associated with subtle long range conformational alterations, further influencing its aggregation behavior. The observation that the fine modulation of A␤ conformation by Ser 8 phosphorylation influences its aggregation is of potential wider interest, especially because A␤ aggregation in vivo usually occurs in various environments such as cellular membrane interfaces where interaction with membrane lipids influences A␤ conformation (40,41).…”