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The binding of monomeric and aggregated variants of 1-83 N-terminal fragment of apolipoprotein A-I with substitution mutations G26R, G26R/W@8, G26R/W@50 and G26R/W@72 to the model lipid membranes composed of phosphatidylcholine and its mixture with cholesterol has been investigated using fluorescent probes pyrene and Laurdan. Examination of pyrene spectral behavior did not reveal any marked influence of apoA-I mutants on the hydrocarbon region of lipid bilayer. In contrast, probing the membrane effects by Laurdan revealed decrease in the probe generalized polarization in the presence of aggregated proteins. suggesting that oligomeric and fibrillar apoA-I species induce increase in hydration degree and reduction of lipid packing density in the membrane interfacial region. These findings may shed light on molecular details of amyloid cytotoxicity. [ABSTRACT FROM AUTHOR]

Highlights: [•] The N-terminal 1–83 residues of apoA-I have a propensity to form amyloid fibrils. [•] ApoA-I 1–43 peptide forms amyloid fibrils with transition to β-sheet-rich structure. [•] The G26R point mutation enhances the fibril formation of apoA-I 1–43 peptide. [•] The extreme N-terminal region plays a crucial role in fibril formation by apoA-I. [ABSTRACT FROM AUTHOR]

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-IIowa) 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 fulllength 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 apoAIIowa: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]