科研之友
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摘要
A mechanistic understanding of A beta aggregation and high-resolution structures of A beta fibrils and oligomers are vital to elucidating relevant details of neurodegeneration in Alzheimer's disease, which will facilitate the rational design of diagnostic and therapeutic protocols. The most detailed and reproducible insights into structure and kinetics have been achieved using A beta peptides produced by recombinant expression, which results in an additional methionine at the N-terminus. While the length of the C-terminus is well established to have a profound impact on the peptide's aggregation propensity, structure, and neurotoxicity, the impact of the N-terminal methionine on the aggregation pathways and structure is unclear. For this reason, we have developed a protocol to produce recombinant A beta(1-42), sans the N-terminal methionine, using an N-terminal small ubiquitin-like modifier-A beta(1-42) fusion protein in reasonable yield, with which we compared aggregation kinetics with A beta(M01-42) containing the additional methionine residue. The data revealed that A beta(1-42) and A beta(M01-42) aggregate with similar rates and by the same mechanism, in which the generation of new aggregates is dominated by secondary nucleation of monomers on the surface of fibrils. We also recorded magic angle spinning nuclear magnetic resonance spectra that demonstrated that excellent spectral resolution is maintained with both A beta(M01-42) and A beta(1-42) and that the chemical shifts are virtually identical in dipolar recoupling experiments that provide information about rigid residues. Collectively, these results indicate that the structure of the fibril core is unaffected by N-terminal methionine. This is consistent with the recent structures of A beta(M01-42) in which M0 is located at the terminus of a disordered 14-amino acid N-terminal tail.
- 出版日期2017-9-12
- 单位MIT
