IVL side-chain methyl groups

Protein Preparation
The IVL labelling scheme produces protein which is uniformly 2H,13C,15N-labelled, except for the Ile, Val and Leu side-chains which are labelled as follows:

IVL labelling pattern

Isoleucine, valine and leucine therefore each contain one methyl group which is 2H12C labelled and one which is 1H13C labelled. In isoleucine it is always the δ1 methyl group which is protonated and 13C labelled and the γ2 methyl group which is deuterated and 12C labelled. For leucine and valine the methyl groups are not stereoselectively labelled. This means that half the valine/leucine in the protein will be 13C and 1H labelled at the γ1/δ1 methyl group and the other half at the γ2/δ2 group. For this reason both valine and leucine methyl groups give rise to peaks in the NMR spectra, but at only about half the intensity of the isoleucine δ1 methyl group.

The protein is produced by expression from bacteria which are grown on minimal medium in D2O using 13C,2H-glucose as the main carbon source and 15NH4Cl as the nitrogen source. One hour prior to induction α-ketobutyrate and α-keto-isovalerate (labelled as shown below) are added to the growth medium and lead to the desired labelling of the Ile and the Val and Leu residues, respectively.

Labelled precursors used for IVL labelling

The labelling scheme was developed in order to improve structure calulcations of large proteins. Large proteins are usually triply labelled and thus only the (mainly backbone) NH groups are 1H-labelled and visible in NMR spectra. NOEs are thus only observed between backbone NH groups which leads to lower quality structures. Tugarinov and Kay developed the IVL labelling scheme, based on the fact, that methyl groups (a) give strong signals due to signal averaging, and (b) are usually found burried in the core – they are thus close to one another and are able to give rise to NOE signals; they will also provide very important structural restraints which are highly complementary to the NH-NH restraints.

See here for variants in which the Ala, Met and Thr methyl groups can also be specifically 1H,13C labelled against a 2H,12C background.

V. Tugarinov and L.E. Kay (2003) J. Am. Chem. Soc. 125 13868-1387. (Link to Article)