Abstract
The interconversion of monomers, oligomers, and amyloid fibrils of the amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer disease. The determination of the kinetics of the individual association and dissociation reactions is hampered by the fact that forward and reverse reactions to/from different aggregation states occur simultaneously. Here, we report the kinetics of dissociation of Aβ monomers from protofibrils, prefibrillar high molecular weight oligomers previously shown to possess pronounced neurotoxicity. An engineered binding protein sequestering specifically monomeric Aβ was employed to follow protofibril dissociation by tryptophan fluorescence, precluding confounding effects of reverse or competing reactions. Aβ protofibril dissociation into monomers follows exponential decay kinetics with a time constant of ∼2 h at 25 °C and an activation energy of 80 kJ/mol, values typical for high affinity biomolecular interactions. This study demonstrates the high kinetic stability of Aβ protofibrils toward dissociation into monomers and supports the delineation of the Aβ folding and assembly energy landscape.
Highlights
Protofibrils of the amyloid- peptide (A) are neurotoxic oligomers implicated in development and progression of Alzheimer disease
ZA3W Is a Tryptophan Fluorescence Probe for Monomeric A—Binding of ZA3 to A is accompanied by a decrease in tyrosine fluorescence [18], presumably due to altered fluorescence properties of Tyr-18 in both ZA3 subunits, which are located at the binding interface
To exploit the higher extinction coefficient, higher quantum yield, and greater environment sensitivity of tryptophan compared with tyrosine, a ZA3 variant termed ZA3W was generated by site-directed mutagenesis of Tyr-18 to Trp-18 (Fig. 1A)
Summary
Protofibrils of the amyloid- peptide (A) are neurotoxic oligomers implicated in development and progression of Alzheimer disease. The interconversion of monomers, oligomers, and amyloid fibrils of the amyloid- peptide (A) has been implicated in the pathogenesis of Alzheimer disease. We report the kinetics of dissociation of A monomers from protofibrils, prefibrillar high molecular weight oligomers previously shown to possess pronounced neurotoxicity. An engineered binding protein sequestering monomeric A was employed to follow protofibril dissociation by tryptophan fluorescence, precluding confounding effects of reverse or competing reactions. This study demonstrates the high kinetic stability of A protofibrils toward dissociation into monomers and supports the delineation of the A folding and assembly energy landscape. The addition of an excess of the tryptophan-containing variant ZA3W to SEC-purified A protofibrils enabled the detection of monomeric A dissociating from protofibrils while preventing the reverse reaction from monomers to protofibrils as well as the reaction to amyloid fibrils. The temperature-dependent kinetic stability of A protofibrils toward dissociation into monomers could be assessed
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