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Apolipoprotein A-I (apoA-I) is an amyloid-forming protein whose amyloidogenic properties are attributed mainly to its N-terminal fragment. Cell membranes are thought to be the primary target for the toxic amyloid aggregates. In the present study Förster resonance energy transfer (FRET) between the membrane fluorescent probe Laurdan as a donor and amyloid-specific dye Thioflavin T (ThT) as an acceptor was employed to explore the interactions of amyloid fibrils from apoA-I variants 1-83/G26R and 1-83/G26R/W@8 with the model membranes composed of phosphatidylcholine and its mixture with cholesterol. The changes in FRET efficiency upon fibril-lipid binding were found to correlate with the extent of protein fibrillization. AFM imaging revealed the presence of two polymorphic states of fibrillar 1-83/G26R/W@8 with the helical and twisted ribbon morphologies. The simulation-based analysis of the experimental FRET profiles provided the arguments in favor of untwisting of fibrillar assemblies upon their interaction with the model membranes. Evidence for the face-on orientation and superficial bilayer location of the membrane-bound fragments of 1-83/G26R/W@8 fibrils was obtained.

Amyloid fibrils are an intriguing class of protein aggregates with distinct physicochemical, structural and morphological properties. They display peculiar membrane-binding behavior, thus adding complexity to the problem of protein-lipid interactions. The consensus that emerged during the past decade is that amyloid cytotoxicity arises from a continuum of cross-β-sheet assemblies including mature fibrils. Based on literature survey and our own data, in this chapter we address several aspects of fibril-lipid interactions, including (i) the effects of amyloid assemblies on molecular organization of lipid bilayer; (ii) competition between fibrillar and monomeric membrane-associating proteins for binding to the lipid surface; and (iii) the effects of lipids on the structural morphology of fibrillar aggregates. To illustrate some of the processes occurring in fibril-lipid systems, we present and analyze fluorescence data reporting on lipid bilayer interactions with fibrillar lysozyme and with the N-terminal 83-residue fragment of amyloidogenic mutant apolipoprotein A-I, 1-83/G26R/W@8. The results help understand possible mechanisms of interaction and mutual remodeling of amyloid fibers and lipid membranes, which may contribute to amyloid cytotoxicity.

The N-terminal 1-83 residues of apolipoprotein A-I (apoA-I) have a strong propensity to form amyloid fibrils, in which the 46-59 segment was reported to aggregate to form amyloid-like fibrils. In this study, we demonstrated that a fragment peptide comprising the extreme N-terminal 1-43 residues strongly forms amyloid fibrils with a transition to β-sheet-rich structure, and that the G26R point mutation enhances the fibril formation of this segment. Our results suggest that in addition to the 46-59 segment, the extreme N-terminal region plays a crucial role in the development of amyloid fibrils by the N-terminal fragment of amyloidogenic apoA-I variants., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Apolipoprotein A-I is amenable to a number of specific mutations associated with hereditary systemic amyloidoses. Amyloidogenic properties of apoA-I are determined mainly by its N-terminal fragment. In the present study Förster resonance energy transfer between tryptophan as a donor and Thioflavin T as an acceptor was employed to obtain structural information on the amyloid fibrils formed by apoA-I variant 1-83/G26R/W@8. Analysis of the dye-fibril binding data provided evidence for the presence of two types of ThT binding sites with similar stoichiometries (bound dye to monomeric protein molar ratio ∼10), but different association constants (∼6 and 0.1μM(-1)) and ThT quantum yields in fibril-associated state (0.08 and 0.05, respectively). A β-strand-loop-β-strand structural model of 1-83/G26R/W@8 apoA-I fibrils has been proposed, with potential ThT binding sites located in the solvent-exposed grooves of the N-terminal β-sheet layer. Reasoning from the expanded FRET analysis allowing for heterogeneity of ThT binding centers and fibril polymorphism, the most probable locations of high- and low-affinity ThT binding sites were attributed to the grooves T16_Y18 and D20_L22, respectively., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A number of naturally occurring mutations of apolipoprotein (apo) A-I, the major protein of HDL, are known to be associated with hereditary amyloidosis and atherosclerosis. Here, we examined the effects of the G26R point mutation in apoA-I (apoA-I(Iowa)) on the structure, stability, and aggregation propensity to form amyloid fibril of full-length apoA-I and the N-terminal fragment of apoA-I. Circular dichroism and fluorescence measurements demonstrated that the G26R mutation destabilizes the N-terminal helix bundle domain of full-length protein, leading to increased hydrophobic surface exposure, whereas it has no effect on the initial structure of the N-terminal 1-83 fragment, which is predominantly a random coil structure. Upon incubation for extended periods at neutral pH, the N-terminal 1-83 variants undergo a conformational change to β-sheet-rich structure with a great increase in thioflavin T fluorescence, whereas no structural change is observed in full-length proteins. Comparison of fibril-forming propensity among substituted mutants at Gly-26 position of 1-83 fragments demonstrated that the G26R mutation enhances the nucleation step of fibril formation, whereas G26K and G26E mutations have small or inhibiting effects on the formation of fibrils. These fibrils of the 1-83 variants have long and straight morphology as revealed by atomic force microscopy and exhibited significant toxicity with HEK293 cells. Our results indicate dual critical roles of the arginine residue at position 26 in apoA-I(Iowa): destabilization of the N-terminal helix bundle structure in full-length protein and enhancement of amyloid fibril formation by the N-terminal 1-83 fragment.