Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson’s disease. Kinetic data and biophysical characterization have shown that the secondary nucleation pathway highly accelerates aggregation via the absorption of monomeric protein on the surface of amyloid fibrils. Here, we used NMR and electron paramagnetic resonance spectroscopy to investigate the interaction of monomeric α-synuclein (α-Syn) with its fibrillar form. We demonstrate that α-Syn monomers interact transiently via their positively charged N terminus with the negatively charged flexible C-terminal ends of the fibrils. These intermolecular interactions reduce intramolecular contacts in monomeric α-Syn, yielding further unfolding of the partially collapsed intrinsically disordered states of α-Syn along with a possible increase in the local concentration of soluble α-Syn and alignment of individual monomers on the fibril surface. Our data indicate that intramolecular unfolding critically contributes to the aggregation kinetics of α-Syn during secondary nucleation.
Finland trityl radical (FTR) shows very attractive EPR spectroscopic properties for a manifold of applications. For most of its applications only one chemically reactive functional group is needed. The presence of three equally reactive carboxyl groups leads to FTR modifications through reactions which give statistical mixtures of 1-fold-, 2-fold-, and 3-fold-modified and unmodified FTR. To avoid the side effects of such a statistical reactionlimited yields and separation challengeswe took a route to FTR-type trityl radicals with scaffold assembly by addition of an aryllithium with one type of substituent to a diarylketone with another type of substituent. This gave the two FTR-type trityl radicals 1 and 2 which carry a combination of the chemically orthogonal groups, carboxyl and triisopropylsilylethynyl. Standard column chromatography was sufficient for product isolation on all stages, whereby polar tagging helped. The EPR spectroscopic properties of the trityl radicals 1 and 2 in ethanol were determined in X and W bands. Their g anisotropy and T 1 and T 2 relaxation times make them spin labels as good as the benchmark FTR. This paper discloses also details on the synthesis of building blocks used for FTR preparation and improved access to the bare FTR scaffold.
A novel statistical analysis of paramagnetic relaxation enhancement (PRE) and paramagnetic relaxation interference (PRI) based nuclear magnetic resonance (NMR) data is proposed based on the computation of correlation matrices. The technique is demonstrated with an example of the intrinsically disordered proteins (IDPs) osteopontin (OPN) and brain acid soluble protein 1 (BASP1). The correlation analysis visualizes in detail the subtleties of conformational averaging in IDPs and highlights the presence of correlated structural fluctuations of individual sub-domains in IDPs.
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