Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins.
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.
Guillaume
G
Mas
Elodie
E
Crublet
Olivier
O
Hamelin
Pierre
P
Gans
Jérôme
J
Boisbouvier
D004268:DNA-Binding Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007553:Isotope Labeling; D008958:Models, Molecular; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D014633:Valine / Q000737:chemistry*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D011506:Proteins; D014633:Valine