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

Understanding the structural and assembly dynamics of the amyloid beta-protein (A beta) 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 beta 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 beta 40) or 42 ( for A beta 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 -> alpha/beta -> beta transition, but substitution-dependent alterations in assembly kinetics and conformer complexity were observed. Tyr(1)-substituted homologues of A beta 40 and A beta 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 beta 40 fibrils. Substitution of A beta 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 beta 40 and A beta 42 as the Tyr substitution position number increased. The ability of a single substitution (Tyr(1)) to alter A beta assembly kinetics and the oligomer frequency distribution suggests that the N terminus is not a benign peptide segment, but rather that A beta 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.