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[NMR paper] NMR-Based Identification of Metabolites in Polar and Non-Polar Extracts of Avian Liver.
Nov 17, 2017 - 12:42 PM - by nmrlearner
nmrlearner's Avatar NMR-Based Identification of Metabolites in Polar and Non-Polar Extracts of Avian Liver.

NMR-Based Identification of Metabolites in Polar and Non-Polar Extracts of Avian Liver.

Metabolites. 2017 Nov 16;7(4):

Authors: Fathi F, Brun A, Rott KH, Falco Cobra P, Tonelli M, Eghbalnia HR, Caviedes-Vidal E, Karasov WH, Markley JL

Abstract
Metabolites present in liver provide important clues regarding the physiological state of an organism. The aim of this work was to evaluate a protocol for high-throughput NMR-based analysis of polar and non-polar metabolites from a small quantity of liver tissue. We extracted the tissue with a methanol/chloroform/water mixture and isolated the polar metabolites from the methanol/water layer and the non-polar metabolites from the chloroform layer. Following drying, we re-solubilized the fractions for analysis with a 600 MHz NMR spectrometer equipped with a 1.7 mm cryogenic probe. In order to evaluate the feasibility of this protocol for metabolomics studies, we analyzed the metabolic profile of livers from house sparrow (Passer domesticus) nestlings raised on two different diets: livers from 10 nestlings raised on a high protein diet (HP) for 4 d and livers from 12 nestlings raised on the HP diet for 3 d and then switched to a high carbohydrate diet (HC) for 1 d. The protocol enabled the detection of 52 polar and nine non-polar metabolites in ¹H NMR spectra of the extracts. We analyzed the lipophilic metabolites by one-way ANOVA to assess statistically significant concentration differences between the two groups. The results of our studies demonstrate that the protocol described here can be exploited for high-throughput screening of small quantities of... [Read More]
0 Replies | 4 Views
[NMR paper] The sign of NMR chemical shift difference as a determinant of the origin of binding selectivity: Elucidation of the position-dependence of phosphorylation in ligands binding to Scribble PDZ1.
Nov 17, 2017 - 12:42 PM - by nmrlearner
nmrlearner's Avatar The sign of NMR chemical shift difference as a determinant of the origin of binding selectivity: Elucidation of the position-dependence of phosphorylation in ligands binding to Scribble PDZ1.

The sign of NMR chemical shift difference as a determinant of the origin of binding selectivity: Elucidation of the position-dependence of phosphorylation in ligands binding to Scribble PDZ1.

Biochemistry. 2017 Nov 16;:

Authors: Sundell G, Vögeli B, Ivarsson Y, Chi C

Abstract
The use of NMR chemical shift perturbation to monitor changes taking place around the binding site of a ligand-protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purposes, amongst others, to determine affinity constants, locate binding surfaces, or differentiate between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Although the directionality of shift changes are often used for detailed analysis of specific cases, the approach has not been adapted in standard chemical shift monitoring. This is surprising as it would not require additional efforts. Here, we demonstrate the importance of the sign of chemical shift difference induced by ligand-protein interaction. We analyze the sign of the 15N/1H shift changes of the PDZ1 domain of Scribble upon interaction with two pairs of phosphorylated and unphosphorylated peptides. We find that detailed differences in the molecular basis of this PDZ-ligand interaction can be obtained from our analysis to which the classical method of combined chemical shift... [Read More]
0 Replies | 4 Views
[NMR paper] Real-Time Observation of the Interaction between Thioflavin T and an Amyloid Protein by Using High-Sensitivity Rheo-NMR.
Nov 17, 2017 - 12:42 PM - by nmrlearner
nmrlearner's Avatar Real-Time Observation of the Interaction between Thioflavin T and an Amyloid Protein by Using High-Sensitivity Rheo-NMR.

Real-Time Observation of the Interaction between Thioflavin T and an Amyloid Protein by Using High-Sensitivity Rheo-NMR.

Int J Mol Sci. 2017 Oct 28;18(11):

Authors: Iwakawa N, Morimoto D, Walinda E, Kawata Y, Shirakawa M, Sugase K

Abstract
Amyloid fibril formation is associated with numerous neurodegenerative diseases. To elucidate the mechanism of fibril formation, the thioflavin T (ThT) fluorescence assay is widely used. ThT is a fluorescent dye that selectively binds to amyloid fibrils and exhibits fluorescence enhancement, which enables quantitative analysis of the fibril formation process. However, the detailed binding mechanism has remained unclear. Here we acquire real-time profiles of fibril formation of superoxide dismutase 1 (SOD1) using high-sensitivity Rheo-NMR spectroscopy and detect weak and strong interactions between ThT and SOD1 fibrils in a time-dependent manner. Real-time information on the interaction between ThT and fibrils will contribute to the understanding of the binding mechanism of ThT to fibrils. In addition, our method provides an alternative way to analyze fibril formation.


PMID: 29143789 [PubMed - in process]



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0 Replies | 4 Views
[NMR paper] Structure of monomeric Interleukin-8 and its interactions with the N-terminal Binding Site-I of CXCR1 by solution NMR spectroscopy.
Nov 17, 2017 - 12:42 PM - by nmrlearner
nmrlearner's Avatar Structure of monomeric Interleukin-8 and its interactions with the N-terminal Binding Site-I of CXCR1 by solution NMR spectroscopy.

Structure of monomeric Interleukin-8 and its interactions with the N-terminal Binding Site-I of CXCR1 by solution NMR spectroscopy.

J Biomol NMR. 2017 Nov 15;:

Authors: Berkamp S, Park SH, De Angelis AA, Marassi FM, Opella SJ

Abstract
The structure of monomeric human chemokine IL-8 (residues 1-66) was determined in aqueous solution by NMR spectroscopy. The structure of the monomer is similar to that of each subunit in the dimeric full-length protein (residues 1-72), with the main differences being the location of the N-loop (residues 10-22) relative to the C-terminal ?-helix and the position of the side chain of phenylalanine 65 near the truncated dimerization interface (residues 67-72). NMR was used to analyze the interactions of monomeric IL-8 (1-66) with ND-CXCR1 (residues 1-38), a soluble polypeptide corresponding to the N-terminal portion of the ligand binding site (Binding Site-I) of the chemokine receptor CXCR1 in aqueous solution, and with 1TM-CXCR1 (residues 1-72), a membrane-associated polypeptide that includes the same N-terminal portion of the binding site, the first trans-membrane helix, and the first intracellular loop of the receptor in nanodiscs. The presence of neither the first transmembrane helix of the receptor nor the lipid bilayer significantly affected the interactions of IL-8 with Binding Site-I of CXCR1.


PMID: 29143165 [PubMed - as supplied by publisher]



... [Read More]
0 Replies | 4 Views
[NMR paper] Using an NMR metabolomics approach to investigate the pathogenicity of amyloid-beta and alpha-synuclein.
Nov 17, 2017 - 12:42 PM - by nmrlearner
nmrlearner's Avatar Using an NMR metabolomics approach to investigate the pathogenicity of amyloid-beta and alpha-synuclein.

Using an NMR metabolomics approach to investigate the pathogenicity of amyloid-beta and alpha-synuclein.

Metabolomics. 2017;13(12):151

Authors: Phelan MM, Caamaño-Gutiérrez E, Gant MS, Grosman RX, Madine J

Abstract
Introduction: The pathogenicity at differing points along the aggregation pathway of many fibril-forming proteins associated with neurodegenerative diseases is unclear. Understanding the effect of different aggregation states of these proteins on cellular processes is essential to enhance understanding of diseases and provide future options for diagnosis and therapeutic intervention.
Objectives: To establish a robust method to probe the metabolic changes of neuronal cells and use it to monitor cellular response to challenge with three amyloidogenic proteins associated with neurodegenerative diseases in different aggregation states.
Method: Neuroblastoma SH-SY5Y cells were employed to design a robust routine system to perform a statistically rigorous NMR metabolomics study into cellular effects of sub-toxic levels of alpha-synuclein, amyloid-beta 40 and amyloid-beta 42 in monomeric, oligomeric and fibrillar conformations.
Results: This investigation developed a rigorous model to monitor intracellular metabolic profiles of neuronal cells through combination of existing methods. This model revealed eight key metabolites that are altered when neuroblastoma cells are challenged with proteins in different aggregation states. Metabolic pathways associated with lipid metabolism, neurotransmission and adaptation to oxidative stress and... [Read More]
0 Replies | 6 Views
Label-free NMR-based dissociation kinetics determination
Nov 16, 2017 - 10:50 AM - by nmrlearner
nmrlearner's Avatar Label-free NMR-based dissociation kinetics determination

Abstract

Understanding the dissociation of molecules is the basis to modulate interactions of biomedical interest. Optimizing drugs for dissociation rates is found to be important for their efficacy, selectivity, and safety. Here, we show an application of the high-power relaxation dispersion (RD) method to the determination of the dissociation rates of weak binding ligands from receptors. The experiment probes proton RD on the ligand and, therefore, avoids the need for any isotopic labeling. The large ligand excess eases the detection significantly. Importantly, the use of large spin-lock fields allows the detection of faster dissociation rates than other relaxation approaches. Moreover, this experimental approach allows to access directly the off-rate of the binding process without the need for analyzing a series of samples with increasing ligand saturation. The validity of the method is shown with small molecule interactions using two macromolecules, bovine serum albumin and tubulin heterodimers.



Source: Journal of Biomolecular NMR
0 Replies | 11 Views
[NMR paper] Phase modulated 2D HSQC-TOCSY for unambiguous assignment of overlapping spin systems
Nov 15, 2017 - 9:02 PM - by nmrlearner
nmrlearner's Avatar Phase modulated 2D HSQC-TOCSY for unambiguous assignment of overlapping spin systems

Publication date: Available online 14 November 2017
Source:Journal of Magnetic Resonance

Author(s): Amrinder Singh, Abhinav Dubey, Satish K. Adiga, Hanudatta S. Atreya

We present a new method that allows one to unambiguously resolve overlapping spin systems often encountered in biomolecular systems such as peptides and proteins or in samples containing a mixture of different molecules such as in metabolomics. We address this problem using the recently proposed phase modulation approach. By evolving the 1H chemical shifts in a conventional two dimensional (2D) HSQC-TOCSY experiment for a fixed delay period, the phase/intensity of set of cross peaks belonging to one spin system are modulated differentially relative to those of its overlapping counterpart, resulting in their discrimination and recognition. The method thus accelerates the process of identification and resonance assignment of individual compounds in complex mixtures. This approach facilitated the assignment of molecules in the embryo culture medium used in human assisted reproductive technology.
Graphical abstract








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0 Replies | 7 Views
Ramped-amplitude NOVEL #DNPNMR
Nov 15, 2017 - 9:02 PM - by nmrlearner
nmrlearner's Avatar From The DNP-NMR Blog:

Ramped-amplitude NOVEL #DNPNMR

p.p1 {margin: 0.0px 0.0px 0.0px 36.0px; text-indent: -36.0px; font: 12.0px Helvetica}
Can, T.V., et al., Ramped-amplitude NOVEL. J. Chem. Phys., 2017. 146(15): p. 154204.


https://doi.org/10.1063/1.4980155


We present a pulsed dynamic nuclear polarization (DNP) study using a ramped-amplitude nuclear orientation via electron spin locking (RA-NOVEL) sequence that utilizes a fast arbitrary waveform generator (AWG) to modulate the microwave pulses together with samples doped with narrow-line radicals such as 1,3-bisdiphenylene-2-phenylallyl (BDPA), sulfonated-BDPA (SA-BDPA), and trityl- OX063. Similar to ramped-amplitude cross polarization in solid-state nuclear magnetic resonance, RA-NOVEL improves the DNP efficiency by a factor of up to 1.6 compared to constant-amplitude NOVEL (CA-NOVEL) but requires a longer mixing time. For example, at mix = 8 s, the DNP efficiency reaches a plateau at a ramp amplitude of 20 MHz for both SA-BDPA and trityl-OX063, regardless of the ramp profile (linear vs. tangent). At shorter mixing times (mix = 0.8 s), we found that the tangent ramp is superior to its linear counterpart and in both cases there exists an optimum ramp size and therefore ramp rate. Our results suggest that RA-NOVEL should be used instead of CA-NOVEL as long as the electronic spin lattice relaxation T1e is sufficiently long and/or the duty cycle of the microwave amplifier is not exceeded. To the best of our knowledge, this is the first example of a time domain... [Read More]
0 Replies | 8 Views
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