Correlation of Backbone Amide and Side-Chain 13C Resonances in Perdeuterated Proteins

✍ Scribed by Frank Löhr; Heinz Rüterjans

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 177 KB
• Volume: 156
• Category: Article
• ISSN: 1090-7807
• DOI: 10.1006/jmre.2002.2539

✦ Synopsis

Side-chain carbon resonance assignments are difficult to obtain for larger proteins. While standard methods require protons for excitation and detection of magnetization, their presence is often unacceptable and often leads to unacceptable relaxation losses at the directly bound carbon sites. In this paper, pulse sequences are presented which provide connectivities between aliphatic sidechain 13 C and amide 1 H and 15 N chemical shifts in fully deuterated, 13 C/ 15 N-enriched proteins. Magnetization either starts off from carbons or from both nitrogens and protons and is passed along the side-chain via 13 C-13 C isotropic mixing. Direct rather than 13 CO-relayed 15 N → 13 C α or 13 C α → 15 N transfer steps allow the detection of intraresidual as well as sequential correlations. To avoid ambiguities between these two types in the three-dimensional version of the experiments, a fourth dimension can be introduced to achieve their separation along a 13 C α frequency axis. The novel methods are demonstrated with the uniformly 2 H/ 13 C/ 15 N labeled 35-kDa protein diisopropylfluorophosphatase from Loligo vulgaris.