3D HCCH3-TOCSY for resonance assignment of methyl-containing side chains in C-13-labeled proteins

Two 3D experiments, (H)CCH3-TOCSY and H(C)CH3-TOCSY, are proposed for reson ance assignment of methyl-containing amino acid side chains. After the init ial proton-carbon INEPT step, during which either carbon or proton chemical shift labeling is achieved (t(1)), the magnetization is spread along the a mino acid side chains by a carbon spin lock. The chemical shifts of methyl carbons are labeled (t(2)) during the following constant time interval. Fin ally the magnetization is transferred, in a reversed INEPT step, to methyl protons for detection (t(3)). 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 c onstant time interval, and an optional removal of CH2, or CH2 and CH, reson ances from the F2F3 planes. The building blocks used in these experiments c an be implemented in a range of heteronuclear experiments focusing on methy l resonances in proteins. The techniques are illustrated using a N-15, C-13 -labeled E93D mutant of Schizosacharomyces pombe phosphoglycerate mutase (2 3.7 kDa). (C) 2000 Academic Press.