Amino Acid Position-specific Contributions to Amyloid β-Protein Oligomerization

Abstract

Understanding the structural and assembly dynamics of the amyloid ␤-protein (A␤) has direct relevance to the development of therapeutic agents for Alzheimer disease. To elucidate these dynamics, we combined scanning amino acid substitution with a method for quantitative determination of the A␤ oligomer frequency distribution, photo-induced cross-linking of unmodified proteins (PICUP), to perform "scanning PICUP." Tyr, a reactive group in PICUP, was substituted at position 1, 10, 20, 30, or 40 (for A␤40) or 42 (for A␤42). The effects of these substitutions were probed using circular dichroism spectroscopy, thioflavin T binding, electron microscopy, PICUP, and mass spectrometry. All peptides displayed a random coil 3 ␣/␤ 3 ␤ transition, but substitution-dependent alterations in assembly kinetics and conformer complexity were observed. Tyr 1 -substituted homologues of A␤40 and A␤42 assembled the slowest and yielded unusual patterns of oligomer bands in gel electrophoresis experiments, suggesting oligomer compaction had occurred. Consistent with this suggestion was the observation of relatively narrow [Tyr 1 ]A␤40 fibrils. Substitution of A␤40 at the C terminus decreased the population conformational complexity and substantially extended the highest order of oligomers observed. This latter effect was observed in both A␤40 and A␤42 as the Tyr substitution position number increased. The ability of a single substitution (Tyr 1 ) to alter A␤ assembly kinetics and the oligomer frequency distribution suggests that the N terminus is not a benign peptide segment, but rather that A␤ conformational dynamics and assembly are affected significantly by the competition between the N and C termini to form a stable complex with the central hydrophobic cluster.Alzheimer disease (AD) 4 is the most common cause of latelife dementia (1) and is estimated to afflict more than 27 million people worldwide (2). An important etiologic hypothesis is that amyloid ␤-protein (A␤) oligomers are the proximate neurotoxins in AD. Substantial in vivo and in vitro evidence supports this hypothesis (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Neurotoxicity studies have shown that A␤ assemblies are potent neurotoxins (5,(13)(14)(15)(16)(17)(18)(19)(20), and the toxicity of some oligomers can be greater than that of the corresponding fibrils (21). Soluble A␤ oligomers inhibit hippocampal long term potentiation (4,5,13,15,17,18,22) and disrupt cognitive function (23). Compounds that bind and disrupt the formation of oligomers have been shown to block the neurotoxicity of A␤ (24,25). Importantly, recent studies in higher vertebrates (dogs) have shown that substantial reduction in amyloid deposits in the absence of decreases in oligomer concentration has little effect on recovery of neurological function (26).Recent studies of A␤ oligomers have sought to correlate oligomer size and biological activity. Oligomers in the supernatants of fibril preparations centrifuged at 100,000 ϫ g caused sustained calcium influx in rat hippocampal neurons, leading to calpain activati...

Globular state in the oligomers formed by Aβ peptides

“…Gel electrophoresis experiments have shown that A␤ peptides exist as monomers or oligomers of the sizes up to tetramer at micromolar concentration. 12,59 It follows from the electron microscopy studies that the dimers and tetramers have overall spherical shape with the diameter D increasing from 18 Å ͑dimer͒ to 220 Å ͑tetramer͒. 12 The experimental size of the dimer is in a good agreement with our estimate of the size of the dimer core ͑D =2R c =22 Å͒.…”

“…At the same time, experiments detect a continuous distribution of A␤ species from monomers to tetramers. 12,59 The likely source of this discrepancy is an elevated A␤ concentration used in our study. The experimental measurements are performed at micromolar concentrations, whereas the A␤ concentration in the simulations is in the millimolar range ͑that constitutes a 10 3 difference in concentrations͒.…”

Converting the Highly Amyloidogenic Human Calcitonin into a Powerful Fibril Inhibitor by Three-dimensional Structure Homology with a Non-amyloidogenic Analogue

“…Most likely an efficient stabilization should lock the molecule in the socalled "␣-basin" (a free energy surface dominated by ␣-helical structures with minima of comparable free energies separated by low barriers (72)), avoiding the migration toward the "␤-basin." Such ␣-helix 3 ␤-sheet conversion going through progressively looser helical segments can be hampered by targeting either the intact ␣-helix with ligand molecules (73) or the structurally independent folding units (turnlike structures) present in the peptide (72,74). The presence of SDS, used as a helix stabilizer, does lengthen the fibrillation time (the lag time of the onset of fibrillation) but does not abolish hCT aggregation.…”

Effects of the English (H6R) and Tottori (D7N) Familial Alzheimer Disease Mutations on Amyloid β-Protein Assembly and Toxicity

“…A turn at Ser 8 -Gly 9 might be particularly important because it can bring the N-terminal quarter of the peptide into contact with the central hydrophobic cluster region. Maji et al (56) proposed that competition between the N and C termini to form a stable complex with the central hydrophobic cluster underlay these effects. For example, a single amino acid substitution, Asp 1 3 Tyr, alters substantially the A␤ assembly kinetics and oligomer size frequency distribution (56).…”

“…Maji et al (56) proposed that competition between the N and C termini to form a stable complex with the central hydrophobic cluster underlay these effects. For example, a single amino acid substitution, Asp 1 3 Tyr, alters substantially the A␤ assembly kinetics and oligomer size frequency distribution (56). The proximity of His 6 and Asp 7 to the putative Ser 8 -Gly 9 turn suggests that the English and Tottori mutations may affect this structural element, and through this effect, alter overall peptide assembly.…”