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 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 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.
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Acknowledgments
We would like to thank Drs. M. Kainosho, P. Macek, M. Plevin, P. Schanda, A. Sivertsen, Mrs I. Ayala and R. Kerfah, as well as Mr. T. Ogden, for stimulating discussions, Dr. D. Marion for help in processing the NMR spectra and Drs. T. Vernet and M. Noirclerc-Savoye for the preparation of the library of mutants. We thank Dr. B. Franzetti for providing clones of TET2. This work used the RoBioMol, high-field NMR and the isotopic labeling facilities at 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 Program FP7/2007-2013 Grant Agreement no. 260887.
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The NMR spectra used to characterize the incorporation of exogenous l-leucine in overexpressed ubiquitin, as well as the table and the spectra corresponding to the assignment of the TET2 valine methyl groups, are available online: http://link.springer.com/journal/volumesAndIssues/10858 (PDF 2499 kb)
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Mas, G., Crublet, E., Hamelin, O. et al. Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins. J Biomol NMR 57, 251–262 (2013). https://doi.org/10.1007/s10858-013-9785-z
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DOI: https://doi.org/10.1007/s10858-013-9785-z