Real-time monitoring of the aggregation of Alzheimer's amyloid-? via1H magic angle spinning NMR spectroscopy.
Ontology highlight
ABSTRACT:
SUBMITTER: Wang J
PROVIDER: S-EPMC5820189 | biostudies-literature | 2018 Feb
REPOSITORIES: biostudies-literature
Publications
Real-time monitoring of the aggregation of Alzheimer's amyloid-β via<sup>1</sup>H magic angle spinning NMR spectroscopy.
Wang Jian J Yamamoto Tomoya T Bai Jia J Cox Sarah J SJ Korshavn Kyle J KJ Monette Martine M Ramamoorthy Ayyalusamy A
Chemical communications (Cambridge, England) 20180201 16
Proton magic-angle-spinning NMR used for real-time analysis of amyloid aggregation reveals that mechanical rotation of Aβ<sub>1-40</sub> monomers increases the rate of formation of aggregates, and that the increasing lag-time with peptide concentration suggests the formation of growth-incompetent species. EGCG's ability to shift off-pathway aggregation is also demonstrated. ...[more]
Similar Datasets
Project description:This article describes the development and testing of the first automatically microfabricated probes to be used in conjunction with the magic angle coil spinning (MACS) NMR technique. NMR spectroscopy is a versatile technique for a large range of applications, but its intrinsically low sensitivity poses significant difficulties in analyzing mass- and volume-limited samples. The combination of microfabrication technology and MACS addresses several well-known NMR issues in a concerted manner for the first time: (i) reproducible wafer-scale fabrication of the first-in-kind on-chip LC microresonator for inductive coupling of the NMR signal and reliable exploitation of MACS capabilities; (ii) improving the sensitivity and the spectral resolution by simultaneous spinning the detection microcoil together with the sample at the "magic angle" of 54.74° with respect to the direction of the magnetic field (magic angle spinning - MAS), accompanied by the wireless signal transmission between the microcoil and the primary circuit of the NMR spectrometer; (iii) given the high spinning rates (tens of kHz) involved in the MAS methodology, the microfabricated inserts exhibit a clear kinematic advantage over their previously demonstrated counterparts due to the inherent capability to produce small radius cylindrical geometries, thus tremendously reducing the mechanical stress and tearing forces on the sample. In order to demonstrate the versatility of the microfabrication technology, we have designed MACS probes for various Larmor frequencies (194, 500 and 700 MHz) testing several samples such as water, Drosophila pupae, adamantane solid and LiCl at different magic angle spinning speeds.
| S-EPMC3423418 | biostudies-literature
Project description:In recent years, exciting developments in instrument technology and experimental methodology have advanced the field of magic-angle spinning (MAS) nuclear magnetic resonance (NMR) to new heights. Contemporary MAS NMR yields atomic-level insights into structure and dynamics of an astounding range of biological systems, many of which cannot be studied by other methods. With the advent of fast MAS, proton detection, and novel pulse sequences, large supramolecular assemblies, such as cytoskeletal proteins and intact viruses, are now accessible for detailed analysis. In this review, we will discuss the current MAS NMR methodologies that enable characterization of complex biomolecular systems and will present examples of applications to several classes of assemblies comprising bacterial and mammalian cytoskeleton as well as human immunodeficiency virus 1 and bacteriophage viruses. The body of work reviewed herein is representative of the recent advancements in the field, with respect to the complexity of the systems studied, the quality of the data, and the significance to the biology.
| S-EPMC5483179 | biostudies-literature
Project description:Several human diseases are associated with the formation of amyloid aggregates, but experimental characterization of these amyloid fibrils and their oligomeric precursors has remained challenging. Experimental and computational analysis of simpler model systems has therefore been necessary, for instance, on the peptide fragment GNNQQNY7?13 of yeast prion protein Sup35p. Expanding on a previous publication, we report here a detailed structural characterization of GNNQQNY fibrils using magic angle spinning (MAS) NMR. On the basis of additional chemical shift assignments we confirm the coexistence of three distinct peptide conformations within the fibrillar samples, as reflected in substantial chemical shift differences. Backbone torsion angle measurements indicate that the basic structure of these coexisting conformers is an extended ?-sheet. We structurally characterize a previously identified localized distortion of the ?-strand backbone specific to one of the conformers. Intermolecular contacts are consistent with each of the conformers being present in its own parallel and in-register sheet. Overall the MAS NMR data indicate a substantial difference between the structure of the fibrillar and crystalline forms of these peptides, with a clearly increased complexity in the GNNQQNY fibril structure. These experimental data can provide guidance for future work, both experimental and theoretical, and provide insights into the distinction between fibril growth and crystal formation.
| S-EPMC3026921 | biostudies-literature
Project description:Viruses, relatively simple pathogens, are able to replicate in many living organisms and to adapt to various environments. Conventional atomic-resolution structural biology techniques, X-ray crystallography and solution NMR spectroscopy provided abundant information on the structures of individual proteins and nucleic acids comprising viruses; however, viral assemblies are not amenable to analysis by these techniques because of their large size, insolubility, and inherent lack of long-range order. In this article, we review the recent advances in magic angle spinning NMR spectroscopy that enabled atomic-resolution analysis of structure and dynamics of large viral systems and give examples of several exciting case studies.
| S-EPMC4413014 | biostudies-literature
Project description:This study reports a first successful demonstration of a single channel proton 3D and 2D high-throughput ultrafast magic angle spinning (MAS) solid-state NMR techniques in an ultra-high magnetic field (1020MHz) NMR spectrometer comprised of HTS/LTS magnet. High spectral resolution is well demonstrated.
| S-EPMC4688097 | biostudies-literature
Project description:Cytochrome P450 (CYP) 3A4 contributes to the metabolism of approximately 50% of commercial drugs by oxidizing a large number of structurally diverse substrates. Like other endoplasmic reticulum-localized P450s, CYP3A4 contains a membrane-anchoring N-terminal helix and a significant number of hydrophobic domains, important for the interaction between CYP3A4 and the membrane. Although the membrane affects specificity of CYP3A4 ligand binding, the structural details of the interaction have not been revealed so far because X-ray crystallography studies are available only for the soluble domain of CYP3A4. Here we report sample preparation and initial magic-angle spinning (MAS) solid-state NMR (SSNMR) of CYP3A4 (Delta3-12) embedded in a nanoscale membrane bilayer, or Nanodisc. The growth protocol yields approximately 2.5 mg of the enzymatically active, uniformly 13C,15N-enriched CYP3A4 from 1 L of growth medium. Polyethylene glycol 3350-precipitated CYP3A4 in Nanodiscs yields spectra of high resolution and sensitivity, consistent with a folded, homogeneous protein. CYP3A4 in Nanodiscs remains enzymatically active throughout the precipitation protocol as monitored by bromocriptine binding. The 13C line widths measured from 13C-13C 2D chemical shift correlation spectra are approximately 0.5 ppm. The secondary structure distribution within several amino acid types determined from 13C chemical shifts is consistent with the ligand-free X-ray structures. These results demonstrate that MAS SSNMR can be performed on Nanodisc-embedded membrane proteins in a folded, active state. The combination of SSNMR and Nanodisc methodologies opens up new possibilities for obtaining structural information on CYP3A4 and other integral membrane proteins with full retention of functionality.
| S-EPMC2571072 | biostudies-literature
Project description:Biomacromolecules that are challenging for the usual structural techniques can be studied with atomic resolution by solid-state NMR spectroscopy. However, the paucity of distance restraints >5 Å, traditionally derived from measurements of magnetic dipole-dipole couplings between protein nuclei, is a major bottleneck that hampers such structure elucidation efforts. Here, we describe a general approach that enables the rapid determination of global protein fold in the solid phase via measurements of nuclear paramagnetic relaxation enhancements (PREs) in several analogues of the protein of interest containing covalently attached paramagnetic tags, without the use of conventional internuclear distance restraints. The method is demonstrated using six cysteine-EDTA-Cu(2+) mutants of the 56-residue B1 immunoglobulin-binding domain of protein G, for which ~230 longitudinal backbone (15)N PREs corresponding to distances of ~10-20 Å were obtained. The mean protein fold determined in this manner agrees with the X-ray structure with a backbone atom root-mean-square deviation of 1.8 Å.
| S-EPMC3335742 | biostudies-literature
Project description:Osteoarthritis (OA) is a degenerative disease of the joints and results in changes in the biochemical composition of cartilage. Previous studies have been undertaken that have used high-resolution NMR spectroscopy to study the biochemical composition of porcine, canine and bovine cartilage. In the present study, high-resolution magical angle spinning (HR-MAS) NMR spectroscopy at 11.7 T has been used to characterize metabolites and detect differences in the spectral signature of human knee articular cartilage from non-OA healthy cadaver knees and samples acquired from severe OA patients at the time of total knee replacement surgery. A statistically significant difference in the alanine (1.47 p.p.m.), N-acetyl (2.04 p.p.m.), choline (3.25 p.p.m.) and glycine (3.55 p.p.m.) metabolite levels was observed between healthy and OA specimens. The results of the present study indicate that a decrease in the intensity of N-acetyl resonance occurs in the later stages of OA. A positive correlation of the N-acetyl levels as measured by (1)H HR-MAS NMR spectroscopy with the total proteoglycan content in the same cartilage specimens as measured by the glycosaminoglycan (GAG) assay was observed. This indicates that N-acetyl can serve as an important bio-marker of OA disease progression. A decrease in the alanine concentration in OA may be attributed to the degradation of the collagen framework with disease progression and eventual loss of the degradation products that are transported from cartilage into the synovial cavity.
| S-EPMC3299852 | biostudies-other
Project description:Amyloid fibrils are self-assembled filamentous structures associated with protein deposition conditions including Alzheimer's disease and the transmissible spongiform encephalopathies. Despite the immense medical importance of amyloid fibrils, no atomic-resolution structures are available for these materials, because the intact fibrils are insoluble and do not form diffraction-quality 3D crystals. Here we report the high-resolution structure of a peptide fragment of the amyloidogenic protein transthyretin, TTR(105-115), in its fibrillar form, determined by magic angle spinning NMR spectroscopy. The structure resolves not only the backbone fold but also the precise conformation of the side chains. Nearly complete (13)C and (15)N resonance assignments for TTR(105-115) formed the basis for the extraction of a set of distance and dihedral angle restraints. A total of 76 self-consistent experimental measurements, including 41 restraints on 19 backbone dihedral angles and 35 (13)C-(15)N distances between 3 and 6 A were obtained from 2D and 3D NMR spectra recorded on three fibril samples uniformly (13)C, (15)N-labeled in consecutive stretches of four amino acids and used to calculate an ensemble of peptide structures. Our results indicate that TTR(105-115) adopts an extended beta-strand conformation in the amyloid fibrils such that both the main- and side-chain torsion angles are close to their optimal values. Moreover, the structure of this peptide in the fibrillar form has a degree of long-range order that is generally associated only with crystalline materials. These findings provide an explanation of the unusual stability and characteristic properties of this form of polypeptide assembly.
| S-EPMC321745 | biostudies-literature
Project description:REDOR-based experiments with simultaneous 1H-13C and 1H-15N dipolar dephasing are explored for investigating intermolecular protein-protein interfaces in complexes formed by a U-13C,15N-labeled protein and its natural abundance binding partner. The application of a double-REDOR filter (dREDOR) results in a complete dephasing of proton magnetization in the U-13C,15N-enriched molecule while the proton magnetization of the unlabeled binding partner is not dephased. This retained proton magnetization is then transferred across the intermolecular interface by 1H-13C or 1H-15N cross polarization, permitting to establish the residues of the U-13C,15N-labeled protein, which constitute the binding interface. To assign the interface residues, this dREDOR-CPMAS element is incorporated as a building block into 13C-13C correlation experiments. We established the validity of this approach on U-13C,15N-histidine and on a structurally characterized complex of dynactin's U-13C,15N-CAP-Gly domain with end-binding protein 1 (EB1). The approach introduced here is broadly applicable to the analysis of intermolecular interfaces when one of the binding partners in a complex cannot be isotopically labeled.
| S-EPMC6258002 | biostudies-literature