The characterization of low-affinity protein complexes is challenging due to their dynamic nature. Here we present a method to stabilize transient protein complexes in vivo by generating a covalent and conformationally flexible bridge between the interaction partners. A highly active pyrrolysyl tRNA synthetase mutant directs the incorporation of unnatural amino acids bearing bromoalkyl moieties (BrCnK) into proteins. We demonstrate for the first time that low-affinity protein complexes between BrCnK-containing proteins and their binding partners can be stabilized in vivo in bacterial and mammalian cells. Using this approach we determined the crystal structure of a transient GDP-bound complex between a small G-protein and its nucleotide exchange factor. We envision that this approach will prove valuable as a general tool for validating and characterizing protein-protein interactions in vitro and in vivo.
Fumarase activity: an in vivo and in vitro biomarker for acute kidney injury
From The DNP-NMR Blog:
Fumarase activity: an in vivo and in vitro biomarker for acute kidney injury
p.p1 {margin: 0.0px 0.0px 0.0px 36.0px; text-indent: -36.0px; font: 12.0px Helvetica}
Nielsen, P.M., et al., Fumarase activity: an in vivo and in vitro biomarker for acute kidney injury. Scientific Reports, 2017. 7: p. 40812.
http://dx.doi.org/10.1038/srep40812
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05-09-2017 12:37 AM
[NMR paper] Fast NMR-Based Determination of the 3D Structure of the Binding Site of Protein-Ligand Complexes with Weak Affinity Binders.
Fast NMR-Based Determination of the 3D Structure of the Binding Site of Protein-Ligand Complexes with Weak Affinity Binders.
Fast NMR-Based Determination of the 3D Structure of the Binding Site of Protein-Ligand Complexes with Weak Affinity Binders.
Angew Chem Int Ed Engl. 2017 Apr 07;:
Authors: Wälti MA, Riek R, Orts J
Abstract
In early drug discovery approaches, screening hits are often weak affinity binders that are difficult to characterize in structural detail, particularly towards obtaining the 3D structure of...
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04-08-2017 10:57 AM
[NMR paper] Orthogonal spin labeling using click chemistry for in vitro and in vivo applications
Orthogonal spin labeling using click chemistry for in vitro and in vivo applications
Publication date: Available online 2 December 2016
Source:Journal of Magnetic Resonance</br>
Author(s): Svetlana Kucher, Sergei Korneev, Swati Tyagi, Ronja Apfelbaum, Dina Grohmann, Edward A. Lemke, Johann P. Klare, Heinz-Jürgen Steinhoff, Daniel Klose</br>
Site-directed spin labeling for EPR- and NMR spectroscopy has mainly been achieved exploiting the specific reactivity of cysteines. For proteins with native cysteines or for in vivo applications, an alternative...
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12-03-2016 02:09 PM
Diels-Alder reactionâ??triggered bioorthogonal protein decaging in living cells - Nature.com
Diels-Alder reactionâ??triggered bioorthogonal protein decaging in living cells - Nature.com
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Diels-Alder reactionâ??triggered bioorthogonal protein decaging in living cells
Nature.com
A generally applicable strategy, however, remains elusive. Herein we describe a small moleculeâ??triggered bioorthogonal protein decaging technique that relies on the inverse electron-demand Diels-Alder reaction for eliminating a chemically caged protein ...
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Online News
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11-04-2014 02:32 PM
[NMR paper] In vivo and in vitro metabolism of a novel ?2-adrenoceptor agonist, trantinterol: metabolites isolation and identification by LC-MS/MS and NMR.
In vivo and in vitro metabolism of a novel ?2-adrenoceptor agonist, trantinterol: metabolites isolation and identification by LC-MS/MS and NMR.
http://www.bionmr.com//www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--production.springer.de-OnlineResources-Logos-springerlink.gif Related Articles In vivo and in vitro metabolism of a novel ?2-adrenoceptor agonist, trantinterol: metabolites isolation and identification by LC-MS/MS and NMR.
Anal Bioanal Chem. 2013 Mar;405(8):2619-34
Authors: Li K, Qin F, Jing L, Li F, Guo X
Abstract
Trantinterol...
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08-15-2013 07:45 PM
Human multiprotein bridging factor 1 and Calmodulin do not interact in vitro as confirmed by NMR spectroscopy and CaM-agarose affinity chromatography.
Human multiprotein bridging factor 1 and Calmodulin do not interact in vitro as confirmed by NMR spectroscopy and CaM-agarose affinity chromatography.
Human multiprotein bridging factor 1 and Calmodulin do not interact in vitro as confirmed by NMR spectroscopy and CaM-agarose affinity chromatography.
Protein Expr Purif. 2011 Jul 14;
Authors: Babini E, Hu X, Parigi G, Vignali M
The human multiprotein bridging factor 1 (hMBF1) has been established in different cellular types to have the role of transcriptional coactivator. It is also reported to be...
[NMR paper] Protein stabilization by compatible solutes. Effect of diglycerol phosphate on the dy
Protein stabilization by compatible solutes. Effect of diglycerol phosphate on the dynamics of Desulfovibrio gigas rubredoxin studied by NMR.
Related Articles Protein stabilization by compatible solutes. Effect of diglycerol phosphate on the dynamics of Desulfovibrio gigas rubredoxin studied by NMR.
Eur J Biochem. 2003 Dec;270(23):4606-14
Authors: Lamosa P, Turner DL, Ventura R, Maycock C, Santos H
Heteronuclear NMR relaxation measurements and hydrogen exchange data have been used to characterize protein dynamics in the presence or absence of...
Proximity-triggered covalent stabilization of low-affinity protein complexes in vitro and in vivo
Linear Mode
