[NMR paper] Purification and Characterization of Recombinant N-Terminally Pyroglutamate-Modified Amyloid-? Variants and Structural Analysis by Solution NMR Spectroscopy.
Purification and Characterization of Recombinant N-Terminally Pyroglutamate-Modified Amyloid-? Variants and Structural Analysis by Solution NMR Spectroscopy.
Related ArticlesPurification and Characterization of Recombinant N-Terminally Pyroglutamate-Modified Amyloid-? Variants and Structural Analysis by Solution NMR Spectroscopy.
PLoS One. 2015;10(10):e0139710
Authors: Dammers C, Gremer L, Neudecker P, Demuth HU, Schwarten M, Willbold D
Abstract
Alzheimer's disease (AD) is the leading cause of dementia in the elderly and is characterized by memory loss and cognitive decline. Pathological hallmark of AD brains are intracellular neurofibrillary tangles and extracellular amyloid plaques. The major component of these plaques is the highly heterogeneous amyloid-? (A?) peptide, varying in length and modification. In recent years pyroglutamate-modified amyloid-? (pEA?) peptides have increasingly moved into the focus since they have been described to be the predominant species of all N-terminally truncated A?. Compared to unmodified A?, pEA? is known to show increased hydrophobicity, higher toxicity, faster aggregation and ?-sheet stabilization and is more resistant to degradation. Nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful method to investigate the conformations of pEA? isoforms in solution and to study peptide/ligand interactions for drug development. However, biophysical characterization of pEA? and comparison to its non-modified variant has so far been seriously hampered by the lack of highly pure recombinant and isotope-enriched protein. Here we present, to our knowledge, for the first time a reproducible protocol for the production of pEA? from a recombinant precursor expressed in E. coli in natural isotope abundance as well as in uniformly [U-15N]- or [U-13C, 15N]-labeled form, with yields of up to 15 mg/l E. coli culture broth. The chemical state of the purified protein was evaluated by RP-HPLC and formation of pyroglutamate was verified by mass spectroscopy. The recombinant pyroglutamate-modified A? peptides showed characteristic sigmoidal aggregation kinetics as monitored by thioflavin-T assays. The quality and quantity of produced pEA?40 and pEA?42 allowed us to perform heteronuclear multidimensional NMR spectroscopy in solution and to sequence-specifically assign the backbone resonances under near-physiological conditions. Our results suggest that the presented method will be useful in obtaining cost-effective high-quality recombinant pEA?40 and pEA?42 for further physiological and biochemical studies.
PMID: 26436664 [PubMed - as supplied by publisher]
[NMR paper] A Facile method for expression and purification of (15)N isotope-labeled human Alzheimer's ?-amyloid peptides from E. coli for NMR-based structural analysis.
A Facile method for expression and purification of (15)N isotope-labeled human Alzheimer's ?-amyloid peptides from E. coli for NMR-based structural analysis.
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Protein Expr Purif. 2015 Jul 28;
Authors: Sharma SC, Armand T, Aurelia Ball K, Chen A, Pelton JG, Wemmer DE, Head-Gordon T
Abstract
Alzheimer's disease (AD) is a progressive neurodegenerative disease...
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[NMR paper] High-resolution NMR characterization of low abundance oligomers of amyloid-? without purification.
High-resolution NMR characterization of low abundance oligomers of amyloid-? without purification.
High-resolution NMR characterization of low abundance oligomers of amyloid-? without purification.
Sci Rep. 2015;5:11811
Authors: Kotler SA, Brender JR, Vivekanandan S, Suzuki Y, Yamamoto K, Monette M, Krishnamoorthy J, Walsh P, Cauble M, Holl MM, Marsh EN, Ramamoorthy A
Abstract
Alzheimer's disease is characterized by the misfolding and self-assembly of the amyloidogenic protein amyloid-? (A?). The aggregation of A? leads to...
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[NMR paper] High Resolution Structural Characterization of A?42 Amyloid Fibrils by MAS NMR.
High Resolution Structural Characterization of A?42 Amyloid Fibrils by MAS NMR.
High Resolution Structural Characterization of A?42 Amyloid Fibrils by MAS NMR.
J Am Chem Soc. 2015 May 22;
Authors: Colvin MT, Silvers R, Frohm B, Su Y, Linse S, Griffin RG
Abstract
The presence of amyloid plaques composed of amyloid beta (A?) fibrils is a hallmark of Alzheimer's disease (AD). The A? peptide is present as several length variants with two common alloforms consisting of 40 and 42 amino acids, denoted A?1-40 and A?1-42, respectively....
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[NMR paper] Structural analysis of the pyroglutamate-modified isoform of the Alzheimer's disease-related amyloid-? using NMR spectroscopy.
Structural analysis of the pyroglutamate-modified isoform of the Alzheimer's disease-related amyloid-? using NMR spectroscopy.
http://www.bionmr.com//www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--media.wiley.com-assets-2250-98-WileyOnlineLibrary-Button_120x27px_FullText.gif Related Articles Structural analysis of the pyroglutamate-modified isoform of the Alzheimer's disease-related amyloid-? using NMR spectroscopy.
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Authors: Sun N, Hartmann R, Lecher J, Stoldt M, Funke SA, Gremer L, Ludwig HH, Demuth HU, Kleinschmidt M,...
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Structural characterization of recombinant human myoglobin isoforms by (1)H and (129)Xe NMR and molecular dynamics simulations.
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Myoglobin (Mb), the main cytosolic oxygen storage/deliver protein, is also known to interact with different small ligands exerting other fundamental physiological roles. In...
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Expression and purification of (15)N- and (13)C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis.
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Protein Expr Purif. 2011 May 27;
Authors: Long F, Cho W, Ishii Y
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Expression, purification and NMR characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor.
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[NMR paper] Overexpression, purification, and characterization of recombinant Ca-ATPase regulator
Overexpression, purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies.
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Authors: Buck B, Zamoon J, Kirby TL, DeSilva TM, Karim C, Thomas D, Veglia G
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Purification and Characterization of Recombinant N-Terminally Pyroglutamate-Modified Amyloid-? Variants and Structural Analysis by Solution NMR Spectroscopy.
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