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Cobalt-based paramagnetic probe to study RNA-protein interactions by NMR
Feb 25, 2017 - 6:21 AM - by nmrlearner
nmrlearner's Avatar Cobalt-based paramagnetic probe to study RNA-protein interactions by NMR

Publication date: Available online 24 February 2017
Source:Journal of Inorganic Biochemistry

Author(s): Leah M. Seebald, Christopher M. DeMott, Srivathsan Ranganathan, Papa Nii Asare Okai, Anastasia Glazunova, Alan Chen, Alexander Shekhtman, Maksim Royzen

Paramagnetic resonance enhancement (PRE) is an NMR technique that allows studying three-dimensional structures of RNA-protein complexes in solution. RNA strands are typically spin labeled using nitroxide reagents, which provide minimal perturbation to the native structure. The current work describes an alternative approach, which is based on a Co2+-based probe that can be covalently attached to RNA in the vicinity of the protein's binding site using ‘click’ chemistry. Similar to nitroxide spin labels, the transition metal based probe is capable of attenuating NMR signal intensities from protein residues localized <40Å away. The extent of attenuation is related to the probe's distance, thus allowing constructing the protein's contact surface map. This new paradigm has been applied to study binding of HIV-1 nucleocapsid protein, NCp7, to a model RNA pentanucleotide.
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Large dose hyperpolarized water with dissolution-DNP at high magnetic field
Feb 25, 2017 - 6:21 AM - by nmrlearner
nmrlearner's Avatar From The DNP-NMR Blog:

Large dose hyperpolarized water with dissolution-DNP at high magnetic field

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Lipsø, K.W., et al., Large dose hyperpolarized water with dissolution-DNP at high magnetic field. J. Magn. Reson., 2017. 274: p. 65-72.


http://dx.doi.org/10.1016/j.jmr.2016.11.008


We demonstrate a method for the preparation of hyperpolarized water by dissolution Dynamic Nuclear Polarization at high magnetic field. Protons were polarized at 6.7 T and 1.1 K to >70% with frequency modulated microwave irradiation at 188G Hz. 97.2 ± 0.7% of the radical was extracted from the sample in the dissolution in a two-phase system. 16 ± 1 mL of 5.0 M 1H in D2O with a polarization of 13.0 ± 0.9% in the liquid state was obtained, corresponding to an enhancement factor of 4000 ± 300 compared to the thermal equilibrium at 9.4 T and 293 K. A longitudinal relaxation time constant of 16 ± 1 s was measured. The sample was polarized and dissolved in a fluid path compatible with clinical polarizers. The volume of hyperpolarized water produced by this method enables angiography and perfusion measurements in large animals, as well as NMR experiments for studies of e.g. proton exchange and polarization transfer to other nuclei.
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Light Microscopy Provides Deep Look into Protein Structure - Laboratory Equipment
Feb 24, 2017 - 2:20 PM - by nmrlearner
nmrlearner's Avatar Light Microscopy Provides Deep Look into Protein Structure - Laboratory Equipment


Laboratory Equipment


Light Microscopy Provides Deep Look into Protein Structure
Laboratory Equipment
Using a new method coined COLD, scientists at the Max Planck Institute for the Science of Light in Erlangen have now visualized protein structures with a resolution of around 5 Ã?. COLD achieves this unprecedented resolution by operating at a ...

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[NMR paper] Determination of glucose exchange rates and permeability of erythrocyte membrane in preeclampsia and subsequent oxidative stress-related protein damage using dynamic-(19)F-NMR.
Feb 23, 2017 - 11:08 AM - by nmrlearner
nmrlearner's Avatar Determination of glucose exchange rates and permeability of erythrocyte membrane in preeclampsia and subsequent oxidative stress-related protein damage using dynamic-(19)F-NMR.

Related Articles Determination of glucose exchange rates and permeability of erythrocyte membrane in preeclampsia and subsequent oxidative stress-related protein damage using dynamic-(19)F-NMR.

J Biomol NMR. 2017 Feb 21;:

Authors: Dickinson E, Arnold JR, Fisher J

Abstract
The cause of the pregnancy condition preeclampsia (PE) is thought to be endothelial dysfunction caused by oxidative stress. As abnormal glucose tolerance has also been associated with PE, we use a fluorinated-mimic of this metabolite to establish whether any oxidative damage to lipids and proteins in the erythrocyte membrane has increased cell membrane permeability. Data were acquired using (19)F Dynamic-NMR (DNMR) to measure exchange of 3-fluoro-3-deoxyglucose (3-FDG) across the membrane of erythrocytes from 10 pregnant women (5 healthy control women, and 5 from women suffering from PE). Magnetisation transfer was measured using the 1D selective inversion and 2D EXSY pulse sequences, over a range of time delays. Integrated intensities from these experiments were used in matrix diagonalisation to estimate the values of the rate constants of exchange and membrane permeability. No significant differences were observed for the rate of exchange of 3-FDG... [Read More]
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[NMR paper] Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses.
Feb 23, 2017 - 11:08 AM - by nmrlearner
nmrlearner's Avatar Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses.

Related Articles Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses.

Nat Commun. 2017 Feb 22;8:14523

Authors: Toyama Y, Kano H, Mase Y, Yokogawa M, Osawa M, Shimada I

Abstract
Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein ?-subunit (G?) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound G? on a microsecond timescale. We show that G? rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.


PMID: 28223697 [PubMed - in process]



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Determination of glucose exchange rates and permeability of erythrocyte membrane in preeclampsia and subsequent oxidative stress-related protein damage using dynamic- 19 F-NMR
Feb 22, 2017 - 6:28 PM - by nmrlearner
nmrlearner's Avatar Determination of glucose exchange rates and permeability of erythrocyte membrane in preeclampsia and subsequent oxidative stress-related protein damage using dynamic- 19 F-NMR

Abstract

The cause of the pregnancy condition preeclampsia (PE) is thought to be endothelial dysfunction caused by oxidative stress. As abnormal glucose tolerance has also been associated with PE, we use a fluorinated-mimic of this metabolite to establish whether any oxidative damage to lipids and proteins in the erythrocyte membrane has increased cell membrane permeability. Data were acquired using 19F Dynamic-NMR (DNMR) to measure exchange of 3-fluoro-3-deoxyglucose (3-FDG) across the membrane of erythrocytes from 10 pregnant women (5 healthy control women, and 5 from women suffering from PE). Magnetisation transfer was measured using the 1D selective inversion and 2D EXSY pulse sequences, over a range of time delays. Integrated intensities from these experiments were used in matrix diagonalisation to estimate the values of the rate constants of exchange and membrane permeability. No significant differences were observed for the rate of exchange of 3-FDG and membrane permeability between healthy pregnant women and those suffering from PE, leading us to conclude that no oxidative damage had occurred at this carrier-protein site in the membrane.



Source: Journal of Biomolecular NMR
0 Replies | 13 Views
[NMR paper] Expression, Purification, and Monitoring of Conformational Changes of hCB2 TMH67H8 in Different Membrane-Mimetic Lipid Mixtures Using Circular Dichroism and NMR Techniques.
Feb 22, 2017 - 6:28 PM - by nmrlearner
nmrlearner's Avatar Expression, Purification, and Monitoring of Conformational Changes of hCB2 TMH67H8 in Different Membrane-Mimetic Lipid Mixtures Using Circular Dichroism and NMR Techniques.

Related Articles Expression, Purification, and Monitoring of Conformational Changes of hCB2 TMH67H8 in Different Membrane-Mimetic Lipid Mixtures Using Circular Dichroism and NMR Techniques.

Membranes (Basel). 2017 Feb 17;7(1):

Authors: Tiburu EK, Zhuang J, Fleischer HN, Arthur PK, Awandare GA

Abstract
This work was intended to develop self-assembly lipids for incorporating G-protein coupled receptors (GPCRs) in order to improve the success rate for nuclear magnetic resonance spectroscopy (NMR) structural elucidation. We hereby report the expression and purification of uniformly (15)N-labeled human cannabinoid receptor-2 domain in insect cell media. The domain was refolded by screening several membrane mimetic environments. Different q ratios of isotropic bicelles were screened for solubilizing transmembrane helix 6, 7 and 8 (TMH67H8). As the concentration of dimyristoylphosphocholine (DMPC) was increased such that the q ratio was between 0.16 and 0.42, there was less crowding in the cross peaks with increasing q ratio. In bicelles of q = 0.42, the maximum number of cross peaks were obtained and the cross peaks were uniformly dispersed. The receptor domain in bicelles beyond q = 0.42 resulted in peak crowding. These... [Read More]
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Theoretical treatment of pulsed Overhauser dynamic nuclear polarization: Consideration of a general periodic pulse sequence #DNPNMR
Feb 22, 2017 - 6:28 PM - by nmrlearner
nmrlearner's Avatar From The DNP-NMR Blog:

Theoretical treatment of pulsed Overhauser dynamic nuclear polarization: Consideration of a general periodic pulse sequence #DNPNMR

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Nasibulov, E.A., et al., Theoretical treatment of pulsed Overhauser dynamic nuclear polarization: Consideration of a general periodic pulse sequence. JETP Letters, 2016. 103(9): p. 582-587.


http://dx.doi.org/10.1134/S0021364016090113


A general theoretical approach to pulsed Overhauser-type dynamic nuclear polarization (DNP) is presented. Dynamic nuclear polarization is a powerful method to create non-thermal polarization of nuclear spins, thereby enhancing their nuclear magnetic resonance signals. The theory presented can treat pulsed microwave irradiation of electron paramagnetic resonance transitions for periodic pulse sequences of general composition. Dynamic nuclear polarization enhancement is analyzed in detail as a function of the microwave pulse length for rectangular pulses and pulses with finite rise time. Characteristic oscillations of the DNP enhancement are found when the pulse-length is stepwise increased, originating from coherent motion of the electron spins driven by the pulses. Experimental low-field DNP data are in very good agreement with this theoretical approach.
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