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 13CH3-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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Acknowledgments
We would like to thank Dr P. Macek, M. Plevin, O. Hamelin, P. Gans, I. Ayala, C. Amero, and A. Favier for stimulating discussions and assistance in sample preparation or analysis. This work used the RobioMol, High-Field NMR, Isotopic Labeling, and Seq3A platforms of the Grenoble Instruct centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02), and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The research leading to these results has received funding from the European Research Council under the European Community’s Seventh Framework Programme FP7/2007-2013 Grant Agreement no. 260887.
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Crublet, E. et al. (2014). A Cost-Effective Protocol for the Parallel Production of Libraries of 13CH3-Specifically Labeled Mutants for NMR Studies of High Molecular Weight Proteins. In: Chen, Y. (eds) Structural Genomics. Methods in Molecular Biology, vol 1091. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-691-7_17
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DOI: https://doi.org/10.1007/978-1-62703-691-7_17
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