Application Note 25
Isotope Labeling of Alanine Methyl Groups on a Deuterated Background for NMR Studies of High-Molecular-Weight Proteins
Chenyun Guo, Raquel Godoy-Ruiz, and Vitali Tugarinov
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742 USA
State-of-the-art isotope-labeling schemes commonly employed for NMR investigations of high-molecular-weight proteins utilize selective incorporation of protons and 13C isotopes into methyl groups of Ileδ1, Leuδ and Valγ side chains in a highly deuterated environment (commonly referred to as “ILV labeling”) providing a large number of high-quality probes for NMR studies of protein structure and dynamics. Robust ILV labeling methods1,2 and strategic location of ILV side chains in the hydrophobic cores of protein structures3,4 have turned ILV labeling into an indispensable tool for NMR studies of large proteins and macromolecular assemblies.5
More than a decade ago, Gardner and Kay pioneered a technique for selective incorporation of protonated Ileδ1 methyl positions into protein molecules.6 Subsequently, selective methyl labeling/protonation methods have been refined and extended to Leuδ and Valγ methyl sites by the Kay group.7,8 The introduction of methyl transverse relaxation optimized spectroscopy9 (methyl-TROSY) has stimulated the development of improved labeling schemes.10,11 Selective labeling of ILV methyl sites in large proteins on a deuterated background in synergy with methyl-TROSY techniques have had a significant impact on NMR studies of structure and dynamics of large protein assemblies up to ~800 kDa in molecular weight.12-15 Nevertheless, the availability of only three ILV probes oftentimes presents a serious limitation for structural and dynamics studies of large proteins. Recent advances in methyl isotope labeling have focused on the extension of the ILV labeling methodology to methyl positions of alanines (Alaβ).
Methyl groups of alanines serve as an attractive extension of the ILV labeling methodology. Ala methyls are located in close proximity to the backbone; their flexibility is therefore reduced compared to other methyl-bearing side chains in proteins. Ala is one of the most frequently encountered residues in protein hydrophobic cores and at molecular interfaces.16 Recently, several reports have focused upon extending the ILV methodology with (selective) labeling of Alaβ methyl positions in large proteins. Isaacson et al. have introduced a labeling strategy for selective incorporation of 13CH3 groups into alanine residues on a deuterated background.17 Specific 13C labeling and protonation of Alaβ methyls to a level of 95% with minimal background labeling (<1%) in minimal D2O-based bacterial medium supplemented with large amounts of selectively 13C-labeled α-deuterated alanine and co-addition of three deuterated compounds: (i) α-ketoisovalerate-D7, (ii) succinate-D4 and (iii) L-isoleucine-D10 has been reported by Boisbouvier and coworkers.18 This labeling protocol has been closely followed for production of {2H; Alaβ-[13CH3]}-labeled malate synthase G19,20 (MSG) – an 82-kDa enzyme containing 73 methyl groups. Alanine is the most abundant residue in MSG comprising 10.1% of the total amino-acid content. Figure 1 shows the methyl-TROSY 1H-13C correlation map of MSG prepared using the protocol described in more detail in Appendix 1.
Read more by downloading the application note.
