A cost-effective protocol for the parallel production of libraries of 13CH3-specifically labeled mutants for NMR studies of high molecular weight proteins. - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Methods in Molecular Biology Année : 2014

A cost-effective protocol for the parallel production of libraries of 13CH3-specifically labeled mutants for NMR studies of high molecular weight proteins.

Résumé

There is increasing interest in applying NMR spectroscopy to the study of large protein assemblies. Development of methyl-specific labeling protocols combined with improved NMR spectroscopy enable nowadays studies of proteins complexes up to 1 MDa. For such large complexes, the major interest lies in obtaining structural, dynamic and interaction information in solution, which requires sequence-specific resonance assignment of NMR signals. While such analysis is quite standard for small proteins, it remains one of the major bottlenecks when the size of the protein increases. Here, we describe implementation and latest improvements of SeSAM, a fast and user-friendly approach for assignment of methyl resonances in large proteins using mutagenesis. We have improved culture medium to boost the production of methyl-specifically labeled proteins, allowing us to perform small-scale parallel production and purification of a library of (13)CH3-specifically labeled mutants. This optimized protocol is illustrated by assignment of Alanine, Isoleucine, and Valine methyl groups of the homododecameric aminopeptidase PhTET2. We estimated that this improved method allows assignment of ca. 100 methyl cross-peaks in 2 weeks, including 4 days of NMR time and less than 2 k€ of isotopic materials.

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Bactériologie
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Dates et versions

hal-01093893 , version 1 (22-06-2021)

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  • HAL Id : hal-01093893 , version 1
  • PUBMED : 24203337

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Elodie Crublet, Rime Kerfah, Guillaume Mas, Marjolaine Noirclerc-Savoye, Violaine Lantez, et al.. A cost-effective protocol for the parallel production of libraries of 13CH3-specifically labeled mutants for NMR studies of high molecular weight proteins.. Methods in Molecular Biology, 2014, pp.229-44. ⟨hal-01093893⟩
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