Publication date: Available online 30 October 2013 Source:Journal of Pharmaceutical and Biomedical Analysis
Author(s): J.M.P. Viéville , S. Charbonnier , P. Eberling , J-P. Starck , M-A. Delsuc
Non covalent grafting of proteins on affinity phases is a very common approach for isolation, purification and re-concentration of tagged proteins. Many biophysical studies are conducted on these grafted proteins (Surface Plasmon Resonance, Quartz Crystal Microbalance, etc.) showing that the integrity and function of the protein is usually maintained. However, NMR studies of such samples were not undertaken so far, due to the broadening observed on this kind of heterogeneous samples. We present here the use of the HR-MAS technology to obtain 2D NMR spectra of the MAGI-1 PDZ2/6 protein domain, C 13 -labeled, tagged with a His-tag and grafted on a Nickel affinity resin. We optimized the C 13 Methyl SOFAST HMQC experiment allowing to important gains in terms of signal-to-noise. The gain comes from the gathering of proton magnetization from the resin material to the protein under study. Several methyl signals from the unstructured C-terminal tail, which is involved in the binding of the PDZ domain to C-terminal peptides of its partners, were observed and measured. The interaction of the bound PDZ domain with cognate peptides was monitored using less than 500 ?g of protein sample. A response proportional to the peptide K d is obtained, indicating that the method can be used to rapidly and efficiently monitor protein-ligand interactions. Graphical abstract
[Question from NMRWiki Q&A forum] Observing protein-ligand interaction using weakly soluble compounds
Observing protein-ligand interaction using weakly soluble compounds
I am trying to setup an experiment using HSQC with titration of a compound to determine the Kd of the protein-ligand interaction. My difficulties have been in the sample preparation/formulation, as these novel compounds are very weakly soluble in aqueous buffers. I must be able to quantify the soluble concentration of compound over ~6-8 points to generate the binding curve (signal intensity/chemical shift vs. ).
Previous observation of this protein by HSQC and other NMR techniques have used a buffer containing 5 mM...
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An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe.
An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe.
An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe.
J Biomol NMR. 2011 Sep 17;
Authors: Saio T, Ogura K, Shimizu K, Yokochi M, Burke TR, Inagaki F
Abstract
A nuclear magnetic resonance-based ligand screening strategy utilizing a paramagnetic lanthanide probe is presented. By fixing a paramagnetic lanthanide ion to a target protein, a pseudo-contact shift (PCS) and a paramagnetic relaxation...
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09-20-2011 03:10 PM
An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe
An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe
Abstract A nuclear magnetic resonance-based ligand screening strategy utilizing a paramagnetic lanthanide probe is presented. By fixing a paramagnetic lanthanide ion to a target protein, a pseudo-contact shift (PCS) and a paramagnetic relaxation enhancement (PRE) can be observed for both the target protein and its bound ligand. Based on PRE and PCS information, the bound ligand is then screened from the compound library and the structure of the ligandâ??protein complex is determined. PRE is an...
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09-20-2011 05:02 AM
[Question from NMRWiki Q&A forum] Tuning probe failed after a dual probe was replaced with a BBI probe
Tuning probe failed after a dual probe was replaced with a BBI probe
We generally use Dual to run 13C and BBI to run 2D. After changed the probe, the command "edhead" was used to set the probe. Put the sample tube, lock the solvent, and then type "atma" to tune the probe. We always do it like this, but now we can not tune the proton after installed the BBI probe (13C is OK). The dip can not be found by "atma", and "atmm" was also not work on forming a dip. What is the most possible reason for this error? How to solve it and avoid it in the future ? Thanks. (Instrument: Bruker 400 MHz,...
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08-23-2011 05:31 PM
Automated NMR Resonance Assignment of Large Proteins for Protein-Ligand Interaction Studies.
Automated NMR Resonance Assignment of Large Proteins for Protein-Ligand Interaction Studies.
Automated NMR Resonance Assignment of Large Proteins for Protein-Ligand Interaction Studies.
J Am Chem Soc. 2010 Dec 16;
Authors: Gossert AD, Hiller S, Ferna?ndez C
The detection and structural characterization of protein-ligand interactions by solution NMR is central to functional biology research as well as to drug discovery. Here we present a robust and highly automated procedure for obtaining the resonance assignments necessary for studies of such...
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12-18-2010 12:00 PM
Automated NMR Resonance Assignment of Large Proteins for Protein-Ligand Interaction Studies
Automated NMR Resonance Assignment of Large Proteins for Protein-Ligand Interaction Studies
Alvar D. Gossert, Sebastian Hiller and Ce?sar Ferna?ndez
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/ja108383x/aop/images/medium/ja-2010-08383x_0004.gif
Journal of the American Chemical Society
DOI: 10.1021/ja108383x
http://feeds.feedburner.com/~ff/acs/jacsat?d=yIl2AUoC8zA
http://feeds.feedburner.com/~r/acs/jacsat/~4/E3PMYeBSCeE
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12-17-2010 12:50 AM
[NMR paper] Biomolecular NMR using a microcoil NMR probe--new technique for the chemical shift as
Biomolecular NMR using a microcoil NMR probe--new technique for the chemical shift assignment of aromatic side chains in proteins.
Related Articles Biomolecular NMR using a microcoil NMR probe--new technique for the chemical shift assignment of aromatic side chains in proteins.
J Am Chem Soc. 2004 May 12;126(18):5873-8
Authors: Peti W, Norcross J, Eldridge G, O'Neil-Johnson M
A specially designed microcoil probe for use in biomolecular NMR spectroscopy is presented. The microcoil probe shows a mass-based sensitivity increase of a minimal...
A new NMR technique to probe Protein-Ligand interaction
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