Thermodynamic phase diagram of amyloid-ß (16-22) peptide.

The aggregation of monomeric amyloid ß protein (Aß) peptide into oligomers and amyloid fibrils in the mammalian brain is associated with Alzheimer's disease. Insight into the thermodynamic stability of the Aß peptide in different polymeric states is fundamental to defining and predicting the aggregation process. Experimental determination of Aß thermodynamic behavior is challenging due to the transient nature of Aß oligomers and the low peptide solubility. Furthermore, quantitative calculation of a thermodynamic phase diagram for a specific peptide requires extremely long computational times. Here, using a coarse-grained protein model, molecular dynamics (MD) simulations are performed to determine an equilibrium concentration and temperature phase diagram for the amyloidogenic peptide fragment Aß16-22 Our results reveal that the only thermodynamically stable phases are the solution phase and the macroscopic fibrillar phase, and that there also exists a hierarchy of metastable phases. The boundary line between the solution phase and fibril phase is found by calculating the temperature-dependent solubility of a macroscopic Aß16-22 fibril consisting of an infinite number of ß-sheet layers. This in silico determination of an equilibrium (solubility) phase diagram for a real amyloid-forming peptide, Aß16-22, over the temperature range of 277-330 K agrees well with fibrillation experiments and transmission electron microscopy (TEM) measurements of the fibril morphologies formed. This in silico approach of predicting peptide solubility is also potentially useful for optimizing biopharmaceutical production and manufacturing nanofiber scaffolds for tissue engineering.

Structural insights into peptide self-assembly using photo-induced crosslinking experiments and discontinuous molecular dynamics.

Determining the structure of the (oligomeric) intermediates that form during the self-assembly of amyloidogenic peptides is challenging because of their heterogeneous and dynamic nature. Thus, there is need for methodology to analyze the underlying molecular structure of these transient species. In this work, a combination of fluorescence quenching, photo-induced crosslinking (PIC) and molecular dynamics simulation was used to study the assembly of a synthetic amyloid-forming peptide, Aß16-22. A PIC amino acid containing a trifluormethyldiazirine (TFMD) group-Fmoc(TFMD)Phe-was incorporated into the sequence (Aß*16-22). Electrospray ionization ion-mobility spectrometry mass-spectrometry (ESI-IMS-MS) analysis of the PIC products confirmed that Aß*16-22 forms assemblies with the monomers arranged as anti-parallel, in-register ß-strands at all time points during the aggregation assay. The assembly process was also monitored separately using fluorescence quenching to profile the fibril assembly reaction. The molecular picture resulting from discontinuous molecule dynamics simulations showed that Aß16-22 assembles through a single-step nucleation into a ß-sheet fibril in agreement with these experimental observations. This study provides detailed structural insights into the Aß16-22 self-assembly processes, paving the way to explore the self-assembly mechanism of larger, more complex peptides, including those whose aggregation is responsible for human disease.

Molecular insights into the surface-catalyzed secondary nucleation of amyloid-ß40 (Aß40) by the peptide fragment Aß16-22.

Understanding the structural mechanism by which proteins and peptides aggregate is crucial, given the role of fibrillar aggregates in debilitating amyloid diseases and bioinspired materials. Yet, this is a major challenge as the assembly involves multiple heterogeneous and transient intermediates. Here, we analyze the co-aggregation of Aß40 and Aß16-22, two widely studied peptide fragments of Aß42 implicated in Alzheimer's disease. We demonstrate that Aß16-22 increases the aggregation rate of Aß40 through a surface-catalyzed secondary nucleation mechanism. Discontinuous molecular dynamics simulations allowed aggregation to be tracked from the initial random coil monomer to the catalysis of nucleation on the fibril surface. Together, the results provide insight into how dynamic interactions between Aß40 monomers/oligomers on the surface of preformed Aß16-22 fibrils nucleate Aß40 amyloid assembly. This new understanding may facilitate development of surfaces designed to enhance or suppress secondary nucleation and hence to control the rates and products of fibril assembly.