Related ArticlesSpecific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins.
J Biomol NMR. 2013 Sep 28;
Authors: Mas G, Crublet E, Hamelin O, Gans P, Boisbouvier J
Abstract
The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D2O medium supplemented with either labeled ?-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d10. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468*kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.
PMID: 24078041 [PubMed - as supplied by publisher]
[NMR paper] A straightforward method for stereospecific assignment of val and leu prochiral methyl groups by solid-state NMR: Scrambling in the [2-(13)C]Glucose labeling scheme.
A straightforward method for stereospecific assignment of val and leu prochiral methyl groups by solid-state NMR: Scrambling in the Glucose labeling scheme.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles A straightforward method for stereospecific assignment of val and leu prochiral methyl groups by solid-state NMR: Scrambling in the Glucose labeling scheme.
J Magn Reson. 2013 Jan 4;228C:45-49
Authors: Lv G, Faßhuber HK, Loquet A, Demers JP, Vijayan V, Giller K, Becker S, Lange A
...
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A straightforward method for stereospecific assignment of val and leu prochiral methyl groups by solid-state NMR: Scrambling in the [2-13C]Glucose labeling scheme
A straightforward method for stereospecific assignment of val and leu prochiral methyl groups by solid-state NMR: Scrambling in the Glucose labeling scheme
Available online 4 January 2013
Publication year: 2013
Source:Journal of Magnetic Resonance</br>
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The unambiguous stereospecific assignment of the prochiral methyl groups in Val and Leu plays an important role in the structural investigation of proteins by NMR. Here, we present a straightforward method for their stereospecific solid-state NMR assignment based on Glucose (Glc) as the sole carbon source during...
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01-08-2013 09:23 AM
Selective editing of Val and Leu methyl groups in high molecular weight protein NMR
Selective editing of Val and Leu methyl groups in high molecular weight protein NMR
Abstract The development of methyl-TROSY approaches and specific 13Câ??1H labeling of Ile, Leu and Val methyl groups in highly deuterated proteins has made it possible to study high molecular weight proteins, either alone or in complexes, using solution nuclear magnetic resonance (NMR) spectroscopy. Here we present 2-dimensional (2D) and 3-dimensional (3D) NMR experiments designed to achieve complete separation of the methyl resonances of Val and Leu, labeled using the same precursor, α-ketoisovalerate...
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05-01-2012 07:06 AM
Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methylâ??methyl nuclear overhauser enhancement spectroscopy
Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methylâ??methyl nuclear overhauser enhancement spectroscopy
Abstract Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to ~1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic...
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09-26-2011 06:42 AM
An optimized isotopic labelling strategy of isoleucine-?(2) methyl groups for solution NMR studies of high molecular weight proteins.
An optimized isotopic labelling strategy of isoleucine-?(2) methyl groups for solution NMR studies of high molecular weight proteins.
An optimized isotopic labelling strategy of isoleucine-?(2) methyl groups for solution NMR studies of high molecular weight proteins.
Chem Commun (Camb). 2011 Jul 26;
Authors: Ayala I, Hamelin O, Amero C, Pessey O, Plevin MJ, Gans P, Boisbouvier J
An efficient synthetic route is proposed to produce 2-hydroxy-2-ethyl-3-oxobutanoate for the specific labelling of Ile methyl-?(2) groups in proteins. The (2)H,...
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07-28-2011 10:51 AM
Alanine Methyl Groups as NMR Probes of Molecular Structure and Dynamics in High-Molecular-Weight Proteins
Alanine Methyl Groups as NMR Probes of Molecular Structure and Dynamics in High-Molecular-Weight Proteins
Raquel Godoy-Ruiz, Chenyun Guo and Vitali Tugarinov
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/ja1083656/aop/images/medium/ja-2010-083656_0009.gif
Journal of the American Chemical Society
DOI: 10.1021/ja1083656
http://feeds.feedburner.com/~ff/acs/jacsat?d=yIl2AUoC8zA
http://feeds.feedburner.com/~r/acs/jacsat/~4/hxZ4cabF688
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12-08-2010 10:04 AM
[NMR paper] Quantitative NMR studies of high molecular weight proteins: application to domain ori
Quantitative NMR studies of high molecular weight proteins: application to domain orientation and ligand binding in the 723 residue enzyme malate synthase G.
Related Articles Quantitative NMR studies of high molecular weight proteins: application to domain orientation and ligand binding in the 723 residue enzyme malate synthase G.
J Mol Biol. 2003 Apr 11;327(5):1121-33
Authors: Tugarinov V, Kay LE
A high-resolution multidimensional NMR study of ligand-binding to Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4...
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11-24-2010 09:01 PM
High Resolution 1H Detected 1H,13C Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective 1H,2H Isotopic Labeling of Methyl Groups
High Resolution <SUP>1</SUP>H Detected <SUP>1</SUP>H,<SUP>13</SUP>C Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective <SUP>1</SUP>H,<SUP>2</SUP>H Isotopic Labeling of Methyl Groups
Vipin Agarwal, Anne Diehl, Nikolai Skrynnikov, and Bernd Reif
J. Am. Chem. Soc.; 2006; 128(39) pp 12620 - 12621;
Abstract:
MAS solid-state NMR experiments applied to biological solids are still hampered by low sensitivity and resolution. In this work, we employ a deuteration scheme in which individual methyl groups are selectively protonated. This labeling scheme...
Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins.
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