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Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in situ protein NMR analysis
Dec 10, 2017 - 1:21 AM - by nmrlearner
nmrlearner's Avatar Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in situ protein NMR analysis

Abstract

Organic synthesis of a ligand with high binding affinities for paramagnetic lanthanide ions is an effective way of generating paramagnetic effects on proteins. These paramagnetic effects manifested in high-resolution NMR spectroscopy are valuable dynamic and structural restraints of proteins and protein‚??ligand complexes. A paramagnetic tag generally contains a metal chelating moiety and a reactive group for protein modification. Herein we report two new DTPA-like tags, 4PS-PyDTTA and 4PS-6M-PyDTTA that can be site-specifically attached to a protein with a stable thioether bond. Both protein-tag adducts form stable lanthanide complexes, of which the binding affinities and paramagnetic tensors are tunable with respect to the 6-methyl group in pyridine. Paramagnetic relaxation enhancement (PRE) effects of Gd(III) complex on protein-tag adducts were evaluated in comparison with pseudocontact shift (PCS), and the results indicated that both 4PS-PyDTTA and 4PS-6M-PyDTTA tags are rigid and present high-quality PREs that are crucially important in elucidation of the dynamics and interactions of proteins and protein-ligand complexes. We also show that these two tags are suitable for in-situ protein NMR analysis.



Source: Journal of Biomolecular NMR
0 Replies | 15 Views
Nanodiscs catch misfolding proteins red-handed - Infosurhoy
Dec 10, 2017 - 1:21 AM - by nmrlearner
nmrlearner's Avatar Nanodiscs catch misfolding proteins red-handed - Infosurhoy



Nanodiscs catch misfolding proteins red-handed
Infosurhoy
‚??We're able to stop the aggregation of the protein in this restricted membrane environment so we can monitor what it looks like before it becomes a mass of fibers.‚?? At this point, the researchers use a technique called nuclear magnetic resonance ...


Read here
0 Replies | 22 Views
[NMR paper] Long-range paramagnetic NMR data can provide a closer look on metal coordination in metalloproteins.
Dec 10, 2017 - 1:21 AM - by nmrlearner
nmrlearner's Avatar Long-range paramagnetic NMR data can provide a closer look on metal coordination in metalloproteins.

Related Articles Long-range paramagnetic NMR data can provide a closer look on metal coordination in metalloproteins.

J Biol Inorg Chem. 2017 Dec 07;:

Authors: Cerofolini L, Staderini T, Giuntini S, Ravera E, Fragai M, Parigi G, Pierattelli R, Luchinat C

Abstract
Paramagnetic NMR data can be profitably incorporated in structural refinement protocols of metalloproteins or metal-substituted proteins, mostly as distance or angle restraints. However, they could in principle provide much more information, because the magnetic susceptibility of a paramagnetic metal ion is largely determined by its coordination sphere. This information can in turn be used to evaluate changes occurring in the coordination sphere of the metal when ligands (e.g.: inhibitors) are bound to the protein. This gives an experimental handle on the molecular structure in the vicinity of the metal which falls in the so-called blind sphere. The magnetic susceptibility anisotropy tensors of cobalt(II) and nickel(II) ions bound to human carbonic anhydrase II in free and inhibited forms have been determined. The change of the magnetic susceptibility anisotropy is directly linked to the binding mode of different ligands in the active site of the enzyme. Indication about the metal coordination sphere in the presence of an inhibitor... [Read More]
0 Replies | 17 Views
[NMR paper] Interaction study between HCV NS5A-D2 and NS5B using 19F NMR.
Dec 10, 2017 - 1:21 AM - by nmrlearner
nmrlearner's Avatar Interaction study between HCV NS5A-D2 and NS5B using 19F NMR.

Related Articles Interaction study between HCV NS5A-D2 and NS5B using 19F NMR.

J Biomol NMR. 2017 Dec 07;:

Authors: Dujardin M, Cantrelle FX, Lippens G, Hanoulle X

Abstract
The non structural protein 5A (NS5A) regulates the replication of the hepatitis C viral RNA through a direct molecular interaction of its domain 2 (NS5A-D2) with the RNA dependent RNA polymerase NS5B. Because of conflicting data in the literature, we study here this molecular interaction using fluorinated versions of the NS5A-D2 protein derived from the JFH1 Hepatitis C Virus strain. Two methods to prepare fluorine-labelled NS5A-D2 involving the biosynthetic incorporation of a 19F-tryptophan using 5-fluoroindole and the posttranslational introduction of fluorine by chemical conjugation of 2-iodo-N-(trifluoromethyl)acetamide with the NS5A-D2 cysteine side chains are presented. The dissociation constants (KD) between NS5A-D2 and NS5B obtained with these two methods are in good agreement, and yield values comparable to those derived previously from a surface plasmon resonance study. We compare benefits and limitations of both labeling methods to study the interaction between an intrinsically disordered protein and a large molecular target by 19F NMR.


PMID: 29218486 [PubMed - as supplied by publisher]



... [Read More]
0 Replies | 19 Views
For Some Chaperones, Stability Comes in Pairs | GEN - Genetic Engineering & Biotechnology News
Dec 09, 2017 - 7:49 AM - by nmrlearner
nmrlearner's Avatar
Genetic Engineering & Biotechnology News


For Some Chaperones, Stability Comes in Pairs | GEN
Genetic Engineering & Biotechnology News
Like an acrobatic duo‚??single proteins lend each other greater stability. [Biozentrum University of Basel]. Chaperone molecules are an important part of protein dynamics for daily cellular function. Misfolded proteins are nonfunctional and can cause ...

and more »
... [Read More]
0 Replies | 24 Views
Protein labeling strategies for liquid-state NMR spectroscopy using cell-free synthesis
Dec 09, 2017 - 7:49 AM - by nmrlearner
nmrlearner's Avatar Protein labeling strategies for liquid-state NMR spectroscopy using cell-free synthesis

Publication date: Available online 7 December 2017
Source:Progress in Nuclear Magnetic Resonance Spectroscopy

Author(s): Beate Hoffmann, Frank LŲhr, Aisha Laguerre, Frank Bernhard, Volker DŲtsch

Preparation of a protein sample for liquid-state nuclear magnetic resonance (NMR) spectroscopy analysis requires optimization of many parameters. This review describes labeling strategies for obtaining assignments of protein resonances. Particular emphasis is placed on the advantages of cell-free protein production, which enables exclusive labeling of the protein of interest, thereby simplifying downstream processing steps and increasing the availability of different labeling strategies for a target protein. Furthermore, proteins can be synthesized in milligram yields, and the open nature of the cell-free system allows the addition of stabilizers, scrambling inhibitors or hydrophobic solubilization environments directly during the protein synthesis, which is especially beneficial for membrane proteins. Selective amino acid labeling of the protein of interest, the possibility of addressing scrambling issues and avoiding the need for labile amino acid precursors have been key factors in enabling the introduction of new assignment strategies based on different labeling schemes as well as on new pulse sequences.... [Read More]
0 Replies | 25 Views
In Situ Characterization of Pharmaceutical Formulations by Dynamic Nuclear Polarization Enhanced MAS NMR #DNPNMR
Dec 09, 2017 - 7:49 AM - by nmrlearner
nmrlearner's Avatar From The DNP-NMR Blog:

In Situ Characterization of Pharmaceutical Formulations by Dynamic Nuclear Polarization Enhanced MAS NMR #DNPNMR

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Ni, Q.Z., et al., In Situ Characterization of Pharmaceutical Formulations by Dynamic Nuclear Polarization Enhanced MAS NMR. The Journal of Physical Chemistry B, 2017. 121(34): p. 8132-8141.


http://dx.doi.org/10.1021/acs.jpcb.7b07213


A principal advantage of magic angle spinning (MAS) NMR spectroscopy lies in its ability to determine molecular structure in a noninvasive and quantitative manner. Accordingly, MAS should be widely applicable to studies of the structure of active pharmaceutical ingredients (API) and formulations. However, the low sensitivity encountered in spectroscopy of natural abundance APIs present at low concentration has limited the success of MAS experiments. Dynamic nuclear polarization (DNP) enhances NMR sensitivity and can be used to circumvent this problem provided that suitable paramagnetic polarizing agent can be incorporated into the system without altering the integrity of solid dosages. Here, we demonstrate that DNP polarizing agents can be added in situ during the preparation of amorphous solid dispersions (ASDs) via spray drying and hot-melt extrusion so that ASDs can be examined during drug development. Specifically, the dependence of DNP enhancement on sample composition, radical concentration, relaxation properties of the API and excipients, types of... [Read More]
0 Replies | 28 Views
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