Abstract
The defining feature of the extensive family of amyloid diseases is the formation of networks of entangled elongated protein fibrils and amorphous aggregates exhibiting crossed β-sheet secondary structure. The time course of amyloid conversion has been studied extensively in vitro with the proteins involved in the neurodegenerative pathology of Parkinson's disease (α-synuclein), Alzheimer's disease (Tau) and Huntington's disease (Huntingtin). Although much is known about the thermodynamics and kinetics of the transition from a soluble, intrinsically disordered monomer to the fibrillar end state, the putative oligomeric intermediates, currently considered to be the major initiators of cellular toxicity, are as yet poorly defined. We have detected and characterized amyloid precursors by monitoring AS aggregation with ESIPT (excited state intramolecular protein transfer) probes, one of which, 7MFE [7-(3-maleimido-N-propanamide)-2-(4-diethyaminophenyl)-3-hydroxychromone], is introduced here and compared with a related compound, 6MFC, used previously. A series of 140 spectra for sparsely labeled AS was acquired during the course of aggregation, and resolved into the relative contributions (spectra, intensities) of discrete molecular species including the monomeric, fibrillar, and ensemble of intermediate forms. Based on these findings, a kinetic scheme was devised to simulate progress curves as a function of key parameters. An essential feature of the model, one not previously invoked in schemes of amyloid aggregation, is the catalysis of molecular fuzziness by discrete colloidal nanoparticles arising spontaneously via monomer condensation upon exposure of AS to ≥ 37 °C.