Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Î?Ï?) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative approach, we used multiple Gd3+-Gd3+ distances measured by double electronâ??electron resonance (DEER) experiments to define the metal position, allowing Î?Ï?-tensor determinations from more robust 5-parameter fits that can be performed with a relatively sparse set of PCSs. Using this approach with the 32Â*kDa E. coli aspartate/glutamate binding protein (DEBP), we demonstrate a structural transition between substrate-bound and substrate-free DEBP, supported by PCSs generated by C3-Tm3+ and C3-Tb3+ tags attached to a genetically encoded p-azidophenylalanine residue. The significance of small PCSs was magnified by considering the difference between the chemical shifts measured with Tb3+ and Tm3+ rather than involving a diamagnetic reference. The integrative sparse data approach developed in this work makes poorly soluble proteins of limited stability amenable to structural studies in solution, without having to rely on cysteine mutations for tag attachment.
[NMR paper] Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy.
Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy.
Related Articles Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy.
J Biomol NMR. 2015 Sep 4;
Authors: Barthelmes D, Gränz M, Barthelmes K, Allen KN, Imperiali B, Prisner T, Schwalbe H
Abstract
We recently engineered encodable lanthanide binding tags (LBTs) into proteins and demonstrated their applicability in Nuclear Magnetic Resonance (NMR)...
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09-06-2015 03:37 PM
Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy
Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy
Abstract
We recently engineered encodable lanthanide binding tags (LBTs) into proteins and demonstrated their applicability in Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography and luminescence studies. Here, we engineered two-loop-LBTs into the model protein interleukin-1β (IL1β) and measured 1H, 15N-pseudocontact shifts (PCSs) by NMR spectroscopy. We determined the Î?Ï?-tensors associated with each Tm3+-loaded...
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09-05-2015 01:55 AM
Integral membrane protein structure determination using pseudocontact shifts
Integral membrane protein structure determination using pseudocontact shifts
Abstract
Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary...
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01-21-2015 08:39 PM
A DOTA-amide lanthanide tag for reliable generation of pseudocontact shifts in protein NMR spectra.
A DOTA-amide lanthanide tag for reliable generation of pseudocontact shifts in protein NMR spectra.
A DOTA-amide lanthanide tag for reliable generation of pseudocontact shifts in protein NMR spectra.
Bioconjug Chem. 2011 Aug 31;
Authors: Graham B, Loh CT, Swarbrick JD, Ung P, Shin J, Yagi H, Jia X, Chhabra S, Barlow N, Pintacuda G, Huber T, Otting G
Abstract
Structural studies of proteins and protein-ligand complexes by nuclear magnetic resonance (NMR) spectroscopy can be greatly enhanced by site-specific attachment of lanthanide ions to...
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09-01-2011 05:20 PM
Proteinâ??protein HADDocking using exclusively pseudocontact shifts
Proteinâ??protein HADDocking using exclusively pseudocontact shifts
<div class="Abstract" lang="en">Abstract <div class="normal">In order to enhance the structure determination process of macromolecular assemblies by NMR, we have implemented long-range pseudocontact shift (PCS) restraints into the data-driven protein docking package HADDOCK. We demonstrate the efficiency of the method on a synthetic, yet realistic case based on the lanthanide-labeled N-terminal ε domain of the E. coli DNA polymerase III (ε186) in complex with the HOT domain. Docking from the bound form of the two...
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06-06-2011 12:53 AM
Generation of Pseudocontact Shifts in Protein NMR Spectra with a Genetically Encoded Cobalt(II)-Binding Amino Acid.
Generation of Pseudocontact Shifts in Protein NMR Spectra with a Genetically Encoded Cobalt(II)-Binding Amino Acid.
Generation of Pseudocontact Shifts in Protein NMR Spectra with a Genetically Encoded Cobalt(II)-Binding Amino Acid.
Angew Chem Int Ed Engl. 2011 Jan 17;50(3):692-4
Authors: Nguyen TH, Ozawa K, Stanton-Cook M, Barrow R, Huber T, Otting G
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01-13-2011 12:00 PM
Generation of Pseudocontact Shifts in Protein NMR Spectra with a Genetically Encoded
Generation of Pseudocontact Shifts in Protein NMR Spectra with a Genetically Encoded Cobalt(II)-Binding Amino Acid.
Related Articles Generation of Pseudocontact Shifts in Protein NMR Spectra with a Genetically Encoded Cobalt(II)-Binding Amino Acid.
Angew Chem Int Ed Engl. 2010 Nov 25;
Authors: Nguyen TH, Ozawa K, Stanton-Cook M, Barrow R, Huber T, Otting G
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11-27-2010 02:45 PM
[NMR paper] Improving the accuracy of NMR structures of large proteins using pseudocontact shifts
Improving the accuracy of NMR structures of large proteins using pseudocontact shifts as long-range restraints.
Related Articles Improving the accuracy of NMR structures of large proteins using pseudocontact shifts as long-range restraints.
J Biomol NMR. 2004 Mar;28(3):205-12
Authors: Gaponenko V, Sarma SP, Altieri AS, Horita DA, Li J, Byrd RA
We demonstrate improved accuracy in protein structure determination for large (>/=30 kDa), deuterated proteins (e.g. STAT4(NT)) via the combination of pseudocontact shifts for amide and methyl protons...
Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags
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