title: Purification and Characterization of Recombinant N-Terminally Pyroglutamate-Modified Amyloid-β Variants and Structural Analysis by Solution NMR Spectroscopy.
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-β (Aβ) peptide, varying in length and modification. In recent years pyroglutamate-modified amyloid-β (pEAβ) peptides have increasingly moved into the focus since they have been described to be the predominant species of all N-terminally truncated Aβ. Compared to unmodified Aβ, pEAβ is known to show increased hydrophobicity, higher toxicity, faster aggregation and β-sheet stabilization and is more resistant to degradation. Nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful method to investigate the conformations of pEAβ isoforms in solution and to study peptide/ligand interactions for drug development. However, biophysical characterization of pEAβ 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 pEAβ 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 Aβ peptides showed characteristic sigmoidal aggregation kinetics as monitored by thioflavin-T assays. The quality and quantity of produced pEAβ40 and pEAβ42 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 pEAβ40 and pEAβ42 for further physiological and biochemical studies.
authors.affiliation: Institute of Complex Systems (ICS-6) Structural Biochemistry, Forschungszentrum Jülich, 52425 Jülich, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Dammers
authors.affiliation: Institute of Complex Systems (ICS-6) Structural Biochemistry, Forschungszentrum Jülich, 52425 Jülich, Germany; Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany.
authors.forename: Lothar
authors.initials: L
authors.lastname: Gremer
authors.affiliation: Institute of Complex Systems (ICS-6) Structural Biochemistry, Forschungszentrum Jülich, 52425 Jülich, Germany; Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Neudecker
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Dep. Molecular Drug Biochemistry and Therapy, 06120 Halle (Saale), Germany.
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Demuth
authors.affiliation: Institute of Complex Systems (ICS-6) Structural Biochemistry, Forschungszentrum Jülich, 52425 Jülich, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Schwarten
authors.affiliation: Institute of Complex Systems (ICS-6) Structural Biochemistry, Forschungszentrum Jülich, 52425 Jülich, Germany; Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Willbold
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000378:metabolism*; D058225:Plaque, Amyloid / Q000378:metabolism; D011487:Protein Conformation; D011761:Pyrrolidonecarboxylic Acid / Q000378:metabolism*
chemical_list: D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D011761:Pyrrolidonecarboxylic Acid