72 research outputs found

    Amyloid Structures from Alzheimer’s Disease Patients

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    Lu and colleagues report the structures of β-amyloid fibrils seeded from the brain extracts of two Alzheimer’s disease patients, a game-changing study that could open new avenues for a structure-based design of diagnostic imaging agents and aggregation inhibiting drugs

    Site-specific perturbations of alpha-synuclein fibril structure by the Parkinson's disease associated mutations A53T and E46K.

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    PMCID: PMC3591419This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Parkinson's disease (PD) is pathologically characterized by the presence of Lewy bodies (LBs) in dopaminergic neurons of the substantia nigra. These intracellular inclusions are largely composed of misfolded α-synuclein (AS), a neuronal protein that is abundant in the vertebrate brain. Point mutations in AS are associated with rare, early-onset forms of PD, although aggregation of the wild-type (WT) protein is observed in the more common sporadic forms of the disease. Here, we employed multidimensional solid-state NMR experiments to assess A53T and E46K mutant fibrils, in comparison to our recent description of WT AS fibrils. We made de novo chemical shift assignments for the mutants, and used these chemical shifts to empirically determine secondary structures. We observe significant perturbations in secondary structure throughout the fibril core for the E46K fibril, while the A53T fibril exhibits more localized perturbations near the mutation site. Overall, these results demonstrate that the secondary structure of A53T has some small differences from the WT and the secondary structure of E46K has significant differences, which may alter the overall structural arrangement of the fibrils

    Automated protein resonance assignments of magic angle spinning solid-state NMR spectra of β1 immunoglobulin binding domain of protein G (GB1)

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    Magic-angle spinning solid-state NMR (MAS SSNMR) represents a fast developing experimental technique with great potential to provide structural and dynamics information for proteins not amenable to other methods. However, few automated analysis tools are currently available for MAS SSNMR. We present a methodology for automating protein resonance assignments of MAS SSNMR spectral data and its application to experimental peak lists of the β1 immunoglobulin binding domain of protein G (GB1) derived from a uniformly 13C- and 15N-labeled sample. This application to the 56 amino acid GB1 produced an overall 84.1% assignment of the N, CO, CA, and CB resonances with no errors using peak lists from NCACX 3D, CANcoCA 3D, and CANCOCX 4D experiments. This proof of concept demonstrates the tractability of this problem

    Solid state nuclear magnetic resonance methodology for biomolecular structure determination

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    Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1999.Includes bibliographical references.Several developments in solid state nuclear magnetic resonance (SSNMR) spectroscopy methods are presented. All studies are performed with magic angle spinning (MAS) and high-power proton decoupling, for optimal sensitivity and resolution. Chemical shift are assigned by multi-dimensional correlation spectroscopy in isotopically enriched molecules ...by Chad Michael Rienstra.Ph.D

    Site-Specific Internal Motions in GB1 Protein Microcrystals Revealed by 3D <sup>2</sup>H–<sup>13</sup>C–<sup>13</sup>C Solid-State NMR Spectroscopy

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    <sup>2</sup>H quadrupolar line shapes deliver rich information about protein dynamics. A newly designed 3D <sup>2</sup>H–<sup>13</sup>C–<sup>13</sup>C solid-state NMR magic angle spinning (MAS) experiment is presented and demonstrated on the microcrystalline β1 immunoglobulin binding domain of protein G (GB1). The implementation of <sup>2</sup>H–<sup>13</sup>C adiabatic rotor-echo-short-pulse-irradiation cross-polarization (RESPIRATION CP) ensures the accuracy of the extracted line shapes and provides enhanced sensitivity relative to conventional CP methods. The 3D <sup>2</sup>H–<sup>13</sup>C–<sup>13</sup>C spectrum reveals <sup>2</sup>H line shapes for 140 resolved aliphatic deuterium sites. Motional-averaged <sup>2</sup>H quadrupolar parameters obtained from the line-shape fitting identify side-chain motions. Restricted side-chain dynamics are observed for a number of polar residues including K13, D22, E27, K31, D36, N37, D46, D47, K50, and E56, which we attribute to the effects of salt bridges and hydrogen bonds. In contrast, we observe significantly enhanced side-chain flexibility for Q2, K4, K10, E15, E19, N35, N40, and E42, due to solvent exposure and low packing density. T11, T16, and T17 side chains exhibit motions with larger amplitudes than other Thr residues due to solvent interactions. The side chains of L5, V54, and V29 are highly rigid because they are packed in the core of the protein. High correlations were demonstrated between GB1 side-chain dynamics and its biological function. Large-amplitude side-chain motions are observed for regions contacting and interacting with immunoglobulin G (IgG). In contrast, rigid side chains are primarily found for residues in the structural core of the protein that are absent from protein binding and interactions

    <sup>1</sup>H‑Detected REDOR with Fast Magic-Angle Spinning of a Deuterated Protein

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    Rotational echo double resonance (REDOR) is a highly successful method for heteronuclear distance determination in biological solid-state NMR, and <sup>1</sup>H detection methods have emerged in recent years as a powerful approach to improving sensitivity and resolution for small sample quantities by utilizing fast magic-angle spinning (>30 kHz) and deuteration strategies. In theory, involving <sup>1</sup>H as one of the spins for measuring REDOR effects can greatly increase the distance measurement range, but few experiments of this type have been reported. Here we introduce a pulse sequence that combines frequency-selective REDOR (FSR) with <sup>1</sup>H detection. We demonstrate this method with applications to samples of uniformly <sup>13</sup>C,<sup>15</sup>N,<sup>2</sup>H-labeled alanine and uniformly <sup>13</sup>C,<sup>2</sup>H,<sup>15</sup>N-labeled GB1 protein, back-exchanged with 30% H<sub>2</sub>O and 70% D<sub>2</sub>O, employing a variety of frequency-selective <sup>13</sup>C pulses to highlight unique spectral features. The resulting, robust REDOR effects provide (1) tools for resonance assignment, (2) restraints of secondary structure, (3) probes of tertiary structure, and (4) approaches to determine the preferred orientation of aromatic rings in the protein core

    2D and 3D 15

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