Real-time in-situ 1H NMR of reactions in peptide solution: preaggregation of amyloid-β fragments prior to fibril formation

In-situ analytical methods are essential for the reliable observation of peptide reactions without perturbation of the system. In this work, a real-time in-situ NMR analysis was performed to gain insight into the initial stage of the aggregation of amyloid-beta (A beta) 8-25 monomers, S(8)GY(10)EVHHQKLVFF(20)AEDVG(25), in solution prior to the fibril formation. NMR chemical shift and intensity changes in combination with the CD spectra revealed no changes in A beta secondary structure, but the presence of soluble, oligomeric intermediates followed by the appearance of insoluble and non-structured aggregates before beta-fibril formation. Molecular views of intermediates and aggregation mechanisms were proposed in comparison with NMR spectral changes in wild-type A beta 8-25 and its two mutants, A21G and E22G. The mutation of just one amino acid modified the aggregation properties of A beta 8-25; it slowed or accelerated the fibril formation by controlling the progress of conversion from monomer to aggregate via a soluble, small oligomer.