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

Abstract

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 side-chain 13C and amide 1H and 15N chemical shifts in fully deuterated, 13C/15N-enriched proteins. Magnetization either starts off from carbons or from both nitrogens and protons and is passed along the side-chain via 13C–13C isotropic mixing. Direct rather than 13CO-relayed 15N→13Cα or 13Cα→15N 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 13Cα frequency axis. The novel methods are demonstrated with the uniformly 2H/13C/15N labeled 35-kDa protein diisopropylfluorophosphatase from Loligo vulgaris.