Abstract
We present the optimization of experimental conditions to yield long, rigid apoferritin protein amyloid fibrils, as well as the corresponding fibrillation pathway. Fibril growth kinetics was followed using atomic force microscopy (AFM), transmission electron microscopy (TEM), dynamic light scattering (DLS), circular dichroism (CD), fourier-transform infrared spectroscopy (FTIR), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Among the morphologies identified, we show that the conditions result in small aggregates, as well as medium and long fibrils. Extended incubation times led to progressive unfolding and hydrolysis of the proteins into very short peptide fragments. AFM, SDS-PAGE, and CD support a universal common fibrillation mechanism in which hydrolyzed fragments play the central role. These collective results provide convincing evidence that protein unfolding and complete hydrolysis of the proteins into very short peptide sequences are essential for the formation of the final apoferritin amyloid-like fibrils.
Highlights
Amyloid aggregates are used by nature in numerous and creative ways, ranging from bacteria to humans
The influence of protein concentration on APO fibril formation was studied by atomic force microscopy (AFM)
We recently reported that ferritin, a key component in the regulation of brain iron homeostasis, forms amyloid fibrils that share common traits with the pathological amyloid fibrils found in Alzheimer’s and Parkinson’s disease
Summary
Amyloid aggregates are used by nature in numerous and creative ways, ranging from bacteria to humans. Amyloid structures originate from a wide range of soluble peptides and proteins, wherein peptide or protein monomers spontaneously self-associate into small oligomers, into supramolecular aggregates, and they form fibrillar structures.[2,11] Peptides and globular proteins are known to possess an intrinsic tendency to convert from their native functional states into insoluble amyloid fibrils.[12,13] It is generally agreed that the process of fibril formation starts from a nucleation site or seed comprising partially unfolded proteins.[11,14,15] Native-state globular proteins, as opposed to smaller pathological peptides, possess a condensed, rigid structure, so their fibrillation occurs from the destabilization of the native structure into partially unfolded conformations via substantial changes in environmental conditions (mainly temperature and pH), which are usually extremely denaturing
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