title: A cost-effective protocol for the parallel production of libraries of 13CH3-specifically labeled mutants for NMR studies of high molecular weight proteins.
abstract: 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.
authors.affiliation: Institut de Biologie Structurale Jean-Pierre Ebel, CEA, Grenoble, France.
authors.forename: Elodie
authors.initials: E
authors.lastname: Crublet
authors.forename: Rime
authors.initials: R
authors.lastname: Kerfah
authors.forename: Guillaume
authors.initials: G
authors.lastname: Mas
authors.forename: Marjolaine
authors.initials: M
authors.lastname: Noirclerc-Savoye
authors.forename: Violaine
authors.initials: V
authors.lastname: Lantez
authors.forename: Thierry
authors.initials: T
authors.lastname: Vernet
authors.forename: Jerome
authors.initials: J
authors.lastname: Boisbouvier
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D015723:Gene Library; D007553:Isotope Labeling; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011506:Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D000596:Amino Acids; D011506:Proteins