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Structural insights into the substrate recognition and reaction specificity of the PLP-dependent fold-type I isoleucine 2-epimerase from Lactobacillus buchneri

Rida Awad, Pierre Gans, Jean-Baptiste Reiser

Biochimie > 2017 > 137 > C > 165-173

The isoleucine 2-epimerase from Lactobacillus buchneri has been previously identified and characterized to catalyze the pyridoxal 5′-phosphate (PLP)-dependent racemization and epimerization of a broad spectrum of nonpolar amino acids from L- to D-form and vice versa, in particular isoleucine. In this study, crystal structures of both native and PLP-complex forms of this racemase are presented at 2...

artykuł

Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins

Rime Kerfah, Michael J Plevin, Remy Sounier, Pierre Gans, więcej

Current Opinion in Structural Biology > 2015 > 32 > Complete > 113-122

Nuclear magnetic resonance (NMR) spectroscopy is a uniquely powerful tool for studying the structure, dynamics and interactions of biomolecules at atomic resolution. In the past 15 years, the development of new isotopic labeling strategies has opened the possibility of exploiting NMR spectroscopy in the study of supra-molecular complexes with molecular weights of up to 1MDa. At the core of these isotopic...

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Scrambling free combinatorial labeling of alanine-β, isoleucine-δ1, leucine-proS and valine-proS methyl groups for the detection of long range NOEs

Rime Kerfah, Michael J. Plevin, Ombeline Pessey, Olivier Hamelin, więcej

Journal of Biomolecular NMR > 2015 > 61 > 1 > 73-82

Specific isotopic labeling of methyl groups in proteins has greatly extended the applicability of solution NMR spectroscopy. Simultaneous labeling of the methyl groups of several different amino acid types can offer a larger number of useful probes that can be used for structural characterisations of challenging proteins. Herein, we propose an improved AILV methyl-labeling protocol in which L and...

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The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif

Rida Awad, Marion Sévajol, Isabel Ayala, Anne Chouquet, więcej

FEBS Open Bio > 2015 > 5 > C > 99-106

Eukaryotic EnguLfment and cell MOtility (ELMO) proteins form an evolutionary conserved family of regulators involved in small GTPase dependent actin remodeling processes that regulates the guanine exchange factor activity of some of the Downstream Of CrK (DOCK) family members. Gathered data strongly suggest that DOCK activation by ELMO and the subsequent signaling result from a subtle balance in the...

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Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins

Guillaume Mas, Elodie Crublet, Olivier Hamelin, Pierre Gans, więcej

Journal of Biomolecular NMR > 2013 > 57 > 3 > 251-262

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 α-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...

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The C-terminal polyproline-containing region of ELMO contributes to an increase in the life-time of the ELMO-DOCK complex

Marion Sévajol, Jean-Baptiste Reiser, Anne Chouquet, Julien Pérard, więcej

Biochimie > 2012 > 94 > 3 > 823-828

The eukaryotic Engulfment and Cell Motility (ELMO) proteins form an evolutionary conserved family of key regulators which play a central role in Rho-dependent biological processes such as engulfment and cell motility/migration. ELMO proteins interact with a subset of Downstream of Crk (DOCK) family members, a new type of guanine exchange factors (GEF) for Rac and cdc42 GTPases. The physiological function...

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¹³ C-Labeled Heparan Sulfate Analogue as a Tool To Study Protein/Heparan Sulfate Interactions by NMR Spectroscopy: Application to the CXCL12α Chemokine

Cédric Laguri, Nicolas Sapay, Jean-Pierre Simorre, Bernhard Brutscher, więcej

Journal of the American Chemical Society > 2011 > 133 > 25 > 9642 - 9645

artykuł

A simple biosynthetic method for stereospecific resonance assignment of prochiral methyl groups in proteins

Michael J. Plevin, Olivier Hamelin, Jérôme Boisbouvier, Pierre Gans

Journal of Biomolecular NMR > 2011 > 49 > 2 > 61-67

A new method for stereospecific assignment of prochiral methyl groups in proteins is presented in which protein samples are produced using U-[¹³C]glucose and subsaturating amounts of 2-[¹³C]methyl-acetolactate. The resulting non-uniform labeling pattern allows proR and proS methyl groups to be easily distinguished by their different phases in a constant-time two-dimensional ¹H-¹³C correlation spectra...

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Stereospecific Isotopic Labeling of Methyl Groups for NMR Spectroscopic Studies of High‐Molecular‐Weight Proteins

Pierre Gans, Olivier Hamelin, Remy Sounier, Isabel Ayala, więcej

Angewandte Chemie > 122 > 11 > 2002 - 2006

Manchmal ist weniger mehr: ¹³C¹H₃‐Methylisotopomere können biosynthetisch mithilfe einer Acetolactatvorstufe spezifisch als pro‐S‐Methylgruppen von Leucin‐ und Valinresten in ansonsten perdeuterierte Proteine eingefügt werden. Durch diese Markierungsstrategie wird die Qualität der NMR‐Spektren großer Proteinassoziationen deutlich verbessert.

artykuł

Innentitelbild: Stereospecific Isotopic Labeling of Methyl Groups for NMR Spectroscopic Studies of High‐Molecular‐Weight Proteins (Angew. Chem. 11/2010)

Pierre Gans, Olivier Hamelin, Remy Sounier, Isabel Ayala, więcej

Angewandte Chemie > 122 > 11 > 1940 - 1940

NMR‐Spektroskopie großer Proteinsysteme gelingt nur nach gezielter Protonierung ausgewählter Positionen vor einem perdeuterierten Hintergrund. In der Zuschrift auf S. 2002 ff. von J. Boisbouvier et al. wird eine Acetolactatvorstufe vorgestellt, um biosynthetisch ¹³C¹H₃‐Gruppen spezifisch in die pro‐S‐Methyl‐Positionen von Leucin und Valin einzuführen. Dieses Markierungsverfahren verbessert die Spektrenqualität...

artykuł

Stereospecific Isotopic Labeling of Methyl Groups for NMR Spectroscopic Studies of High‐Molecular‐Weight Proteins

Pierre Gans, Olivier Hamelin, Remy Sounier, Isabel Ayala, więcej

Angewandte Chemie International Edition > 49 > 11 > 1958 - 1962

Sometimes less is more: [¹³C¹H₃]methyl isotopomers can be biosynthetically incorporated specifically into the pro‐S methyl groups of leucine and valine residues in large protein assemblies within a perdeuterated background by using an acetolactate precursor. This stereospecific labeling strategy considerably enhances NMR spectra for large protein assemblies.

artykuł

Inside Cover: Stereospecific Isotopic Labeling of Methyl Groups for NMR Spectroscopic Studies of High‐Molecular‐Weight Proteins (Angew. Chem. Int. Ed. 11/2010)

Pierre Gans, Olivier Hamelin, Remy Sounier, Isabel Ayala, więcej

Angewandte Chemie International Edition > 49 > 11 > 1896 - 1896

NMR spectroscopy of large protein assemblies necessitates targeted protonation of specific sites within a perdeuterated background. In their Communication on page 1958 ff., J. Boisbouvier et al. outline the use of an acetolactate precursor for the specific biosynthetic incorporation of ¹³C¹H₃ into the pro‐S methyl groups of leucine and valine. This labeling strategy enhances spectral quality and provides...

artykuł

An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein

Isabel Ayala, Remy Sounier, Nathalie Usé, Pierre Gans, więcej

Journal of Biomolecular NMR > 2009 > 43 > 2 > 111-119

A strategy for the introduction of (¹H,¹³C-methyl)-alanine into perdeuterated proteins is described. Specific protonation of alanine methyl groups to a level of 95% can be achieved by overexpressing proteins in M9/D₂O based bacterial growth medium supplemented with 800 mg/l of 2-[²H], 3-[¹³C] l-alanine. However, though simple, this approach results in undesired, non-specific background labeling due...

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Engineering functional artificial hybrid proteins between poplar peroxiredoxin II and glutaredoxin or thioredoxin

Nicolas Rouhier, Filipe Gama, Gunnar Wingsle, Eric Gelhaye, więcej

Biochemical and Biophysical Research Communications > 2006 > 341 > 4 > 1300-1308

The existence of natural peroxiredoxin–glutaredoxin hybrid enzymes in several bacteria is in line with previous findings indicating that poplar peroxiredoxin II can use glutaredoxin as an electron donor. This peroxiredoxin remains however unique since it also uses thioredoxin with a quite good efficiency. Based on the existing fusions, we have created artificial enzymes containing a poplar peroxiredoxin...

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Overexpression and purification of Pyrococcus abyssi phosphopantetheine adenylyltransferase from an optimized synthetic gene for NMR studies

Monika Nálezková, Arjan de Groot, Marcus Graf, Pierre Gans, więcej

Protein Expression and Purification > 2005 > 39 > 2 > 296-306

Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme that catalyses a rate-limiting step in coenzyme A (CoA) biosynthesis in all organisms. This study was conducted to obtain a high amount of pure, soluble, and stable PPAT from the hyperthermophilic archaeon Pyrococcus abyssi with the aim of investigating its structural characterization by NMR. Production of this enzyme from its natural...

artykuł

Solution Structural Study of BlaI: Implications for the Repression of Genes Involved in β-Lactam Antibiotic Resistance

Hélène Van Melckebeke, Christelle Vreuls, Pierre Gans, Patrice Filée, więcej

Journal of Molecular Biology > 2003 > 333 > 4 > 711-720

β-Lactamase and penicillin-binding protein PBP2' mediate staphylococcal resistance to β-lactam antibiotics, which are otherwise highly clinically effective. Two repressors (BlaI and MecI) regulate expression of these inducible proteins. Here, we present the first solution structure of the 82 amino acid residue DNA-binding domain of Bacillus licheniformis BlaI which is very similar in primary sequence...