Methyl Isotope-Labeling of Proteins from NMR-Bio

Methyl groups are ideal probes for studying protein structure and function by NMR. Choose among kits for isotope labeling of a single type or multiple types of methyl groups.

Kits for Methyl Group Assignments

Assign specific amino acid methyl groups separately or simultaneously using kits that connect region- or stereo- specific labeled 13CH3 groups to the 15N/13C backbone nuclei via a linear 13C spin system.

Optimized labeling of Ile, Leu, and Val for assignment

Figure 1. Optimized labeling of Ile, Leu, and Val for assignment. Protein was produced in U-[2H,13C,15N] M9 media supplemented with TLAM-Iγ2LVproR-U-[13C] kit.1,2

Kits for Solid-state NMR studies

Achieve region and stereospecific labeling of Ile, Leu, Val, and Ala with 13CHD2 isotopomers. Acquire high-resolution solution and solid-state 1H, 13C spectra to investigate precise dynamics.

Kits for Large Protein Labeling

Investigate protein assemblies with molecular weight up to 1 MDa.

a. Label one type of methyl group or combine kits to label multiple types.

Use a SLAM kit to achieve high incorporation of 13CH3 isotopomer on a single type of methyl group. Use in perdeuterated minimal media without detectable scrambling.

Reduce spectral overlap using combinations of single labeling (SLAM) kits and simultaneously label different types of methyl probes that are well separated in Methyl-TROSY spectra.

Kits for Large Protein Labeling

Figure 2. Kits for Large Protein Labeling

b. Detect long-range nOes or filter inter-molecular nOes in supramolecular assemblies

Extract precise and long-range nOe distant restraints between methyl probes in perdeuterated proteins. Or, filter intra- and inter- molecular nOes and get more precise structural information to refine your homo-oligomeric protein structure.

Combinational Labeling Kits for Long-Range nOes Detection

Solutions for the simultaneous isotope labeling of combinations of Ala, Ile, Leu, Met, Thr, and Val methyl groups

NMR-Bio kits include region-specific and/or stereo-specific labeling of Ile, Leu, and Val residues. Kits are provided with precise protocols extensively tested in vivo to ensure optimal incorporation in targeted methyl groups without detectable scrambling in other positions.

The kits are specifically deuterated and supplied frozen and ready to use. All kits are calibrated for addition into deuterated M9 culture medium prior to induction. These kits incorporate 13CH3 or 13CHD­2 isotopomers into selected protein methyl groups. Protocols are provided with each kit, and each kit is designed for 1 L of culture.

Detection of long-range nOes between methyl probes distant

Figure 3. Detection of long-range nOes between methyl probes distant by up to 10 Å in large proteins and complexes.1,3

Materials
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References

1.
Kerfah R, Plevin MJ, Sounier R, Gans P, Boisbouvier J. 2015. Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins. Current Opinion in Structural Biology. 32113-122. http://dx.doi.org/10.1016/j.sbi.2015.03.009
2.
Kerfah R, Hamelin O, Boisbouvier J, Marion D. 2015. CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample. J Biomol NMR. 63(4):389-402. http://dx.doi.org/10.1007/s10858-015-9998-4
3.
Kerfah R, Plevin MJ, Pessey O, Hamelin O, Gans P, Boisbouvier J. 2015. Scrambling free combinatorial labeling of alanine-?, isoleucine-?1, leucine-proS and valine-proS methyl groups for the detection of long range NOEs. J Biomol NMR. 61(1):73-82. http://dx.doi.org/10.1007/s10858-014-9887-2