3D HCCH3-TOCSY for Resonance Assignment of Methyl-Containing Side Chains in C-Labeled Proteins

Two 3D experiments, (H)CCH₃-TOCSY and H(C)CH₃-TOCSY, are proposed for resonance assignment of methyl-containing amino acid side chains. After the initial proton–carbon INEPT step, during which either carbon or proton chemical shift labeling is achieved (t₁), the magnetization is spread along the amino acid side chains by a carbon spin lock. The chemical shifts of methyl carbons are labeled (t₂) during the following constant time interval. Finally the magnetization is transferred, in a reversed INEPT step, to methyl protons for detection (t₃). The proposed experiments are characterized by high digital resolution in the methyl carbon dimension (t_2max = 28.6 ms), optimum sensitivity due to the use of proton decoupling during the long constant time interval, and an optional removal of CH₂, or CH₂ and CH, resonances from the F₂F₃ planes. The building blocks used in these experiments can be implemented in a range of heteronuclear experiments focusing on methyl resonances in proteins. The techniques are illustrated using a ¹⁵N, ¹³C-labeled E93D mutant of Schizosacharomyces pombe phosphoglycerate mutase (23.7 kDa).