3D HCCH3-TOCSY for Resonance Assignment of Methyl-Containing Side Chains in 13C-Labeled Proteins

✍ Scribed by Dušan Uhrı́n; Stanislava Uhrı́nová; Claire Leadbeater; Jacqueline Nairn; Nicholas C Price; Paul N Barlow

• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 130 KB
• Volume: 142
• Category: Article
• ISSN: 1090-7807
• DOI: 10.1006/jmre.1999.1951

✦ Synopsis

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).