Preparation of Amyloid Fibrils for Magic-Angle Spinning Solid-State NMR Spectroscopy

Tuttle, Marcus D.; Courtney, Joseph M.; Barclay, Alexander M.; Rienstra, Chad M.

doi:10.1007/978-1-4939-2978-8_11

Cited by 8 publications

(6 citation statements)

References 14 publications

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“…We determined a 13 C linewidth on the order of 250 Hz to 300 Hz, which indicates a high degree of structural heterogeneity in the precipitate. By contrast, structured amyloid aggregates display 13 C linewidths of approximately 100 Hz 53 . Still, we obtained a spectrum, in which we could assign the spin systems of nine amino acids ( Fig.…”

Section: Resultsmentioning

confidence: 95%

Epigallocatechin-3-gallate preferentially induces aggregation of amyloidogenic immunoglobulin light chains

Hora

Carballo-Pacheco

Weber

et al. 2017

Sci Rep

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Antibody light chain amyloidosis is a rare disease caused by fibril formation of secreted immunoglobulin light chains (LCs). The huge variety of antibody sequences puts a serious challenge to drug discovery. The green tea polyphenol epigallocatechin-3-gallate (EGCG) is known to interfere with fibril formation in general. Here we present solution- and solid-state NMR studies as well as MD simulations to characterise the interaction of EGCG with LC variable domains. We identified two distinct EGCG binding sites, both of which include a proline as an important recognition element. The binding sites were confirmed by site-directed mutagenesis and solid-state NMR analysis. The EGCG-induced protein complexes are unstructured. We propose a general mechanistic model for EGCG binding to a conserved site in LCs. We find that EGCG reacts selectively with amyloidogenic mutants. This makes this compound a promising lead structure, that can handle the immense sequence variability of antibody LCs.

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Section: Resultsmentioning

confidence: 95%

Epigallocatechin-3-gallate preferentially induces aggregation of amyloidogenic immunoglobulin light chains

Hora

Carballo-Pacheco

Weber

et al. 2017

Sci Rep

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“…A number of similar studies4243444546 have been reported using purified recombinant proteins. Typically, WT α-synuclein fibrils, as detected by ThT binding assays, are obtained by incubating monomeric WT α-synuclein at high micromolar concentrations (50–1000 μM) at 37 °C, upon agitation for 1–5 days424346. Fibrils from α-synuclein mutants are formed in almost the same conditions, with a 2–3 fold increase or decrease in maturation time.…”

Section: Discussionmentioning

confidence: 94%

Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

Sierecki

Giles

Bowden

et al. 2016

Sci Rep

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Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer’s and Parkinson’s disease. Parkinson’s disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson’s disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.

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“…Expression and purification of α-syn were developed as described in the previous study. 27 The recombinant α-syn was dissolved in 20 mM Trisbuffered saline (TBS) buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl) and sonicated in an ice bath for 10 min, then centrifuged at 12 000g for 20 min at 4 °C, 80% of the supernatant was collected to remove the precipitation. The concentration of α-syn was determined by NanoDrop 2000 fluorometer (Thermo Fisher Scientific, MA, USA).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Brazilin Inhibits α-Synuclein Fibrillogenesis, Disrupts Mature Fibrils, and Protects against Amyloid-Induced Cytotoxicity

Liu¹,

Wang

Sang

et al. 2019

J. Agric. Food Chem.

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The inhibitory effect of brazilin against α-synuclein (α-syn) fibrillogenesis, disruption effect against mature fibrils, and the following cytotoxicity were examined by systematical biochemical, biophysical, cellular biological, and molecular simulation experiments. It is found that brazilin inhibited α-syn fibrillogenesis and disrupted the performed fibrils with a concentration-dependent manner. Moreover, cellular experimental data showed that brazilin effectively reduced the cytotoxicity induced by α-syn aggregates. Finally, molecular dynamics simulations were performed to explore the interactions between brazilin and α-syn pentamer. It is found that brazilin directly interacts with α-syn pentamer, and the hydrophobic interactions are favorable for brazilin binding with the α-syn pentamer, while the electrostatic part provides adverse effects. Three binding regions were identified to inhibit α-syn fibrillogenesis or disrupt the preformed aggregates. Furthermore, six important residues (i.e., G51, V52, A53, E61, V66, and K80) of α-syn were also identified. We expected that brazilin is an effective agent against α-syn fibrillogenesis and associated cytotoxicity.

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Preparation of Amyloid Fibrils for Magic-Angle Spinning Solid-State NMR Spectroscopy

Cited by 8 publications

References 14 publications

Epigallocatechin-3-gallate preferentially induces aggregation of amyloidogenic immunoglobulin light chains

Epigallocatechin-3-gallate preferentially induces aggregation of amyloidogenic immunoglobulin light chains

Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

Brazilin Inhibits α-Synuclein Fibrillogenesis, Disrupts Mature Fibrils, and Protects against Amyloid-Induced Cytotoxicity

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