Two 3D experiments, (H)CCH(3)-TOCSY and H(C)CH(3)-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 (t(1)), the magnetization is spread along the amino acid side chains by a carbon spin lock. The chemical shifts of methyl carbons are labeled (t(2)) during the following constant time interval. Finally 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 constant time interval, and an optional removal of CH(2), or CH(2) and CH, resonances from the F(2)F(3) 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 (15)N, (13)C-labeled E93D mutant of Schizosacharomyces pombe phosphoglycerate mutase (23.7 kDa).

• PMID: 10648145
• DOI full text
• PubMed
• PubTator

Reference Type

Journal Article | Research Support, Non-U.S. Gov't

Authors

Uhrín D, Uhrínová S, Leadbeater C, Nairn J, Price NC, Barlow PN

Primary Lit For

Additional Lit For

Review For