75 research outputs found
Amyloid Structures from Alzheimer’s Disease Patients
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
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Amphotericin forms an extramembranous and fungicidal sterol sponge.
For over 50 years, amphotericin has remained the powerful but highly toxic last line of defense in treating life-threatening fungal infections in humans with minimal development of microbial resistance. Understanding how this small molecule kills yeast is thus critical for guiding development of derivatives with an improved therapeutic index and other resistance-refractory antimicrobial agents. In the widely accepted ion channel model for its mechanism of cytocidal action, amphotericin forms aggregates inside lipid bilayers that permeabilize and kill cells. In contrast, we report that amphotericin exists primarily in the form of large, extramembranous aggregates that kill yeast by extracting ergosterol from lipid bilayers. These findings reveal that extraction of a polyfunctional lipid underlies the resistance-refractory antimicrobial action of amphotericin and suggests a roadmap for separating its cytocidal and membrane-permeabilizing activities. This new mechanistic understanding is also guiding development of what are to our knowledge the first derivatives of amphotericin that kill yeast but not human cells
Site-specific perturbations of alpha-synuclein fibril structure by the Parkinson's disease associated mutations A53T and E46K.
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)
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
Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications
The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form
Solid state nuclear magnetic resonance methodology for biomolecular structure determination
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 2H13C13C Solid-State NMR Spectroscopy
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
<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
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
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