A novel strategy for the assignment of side-chain resonances in completely deuterated large proteins using 13C spectroscopy

被引:85
作者
Eletsky, A
Moreira, O
Kovacs, H
Pervushin, K [1 ]
机构
[1] ETH Honggerberg, Swiss Fed Inst Technol, Phys Chem Lab, CH-8093 Zurich, Switzerland
[2] Bruker BioSpin AG, CH-8117 Fallanden, Switzerland
关键词
D O I
10.1023/A:1023572320699
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The assignment of the aliphatic C-13 resonances of trimeric Bacillus Subtilis chorismate mutase, a protein with a molecular mass of 44 kDa, consisting of three 127-residue monomers is presented by use of two-dimensional (2D) C-13-start and C-13-observe NMR experiments. These experiments start with C-13 excitation and end with C-13 observation while relying on the long transverse relaxation times of C-13 spins in uniformly deuterated and C-13, N-15 labeled large proteins. Gains in sensitivity are achieved by the use of a paramagnetic relaxation enhancement agent to reduce C-13 T-1 relaxation times with little effect on C-13 T-2 relaxation times. Such 2D C-13-only NMR experiments circumvent problems associated with the application of conventional experiments for side-chain assignment to proteins of larger sizes, for instance, the absence or low concentration of the side-chain H-1 spins, the transfer of the side-chain spin polarization to the H-1(N) spins for signal acquisition, or the necessity of a quantitative reprotonation of the methyl moieties in the otherwise fully deuterated side-chains. We demonstrate that having obtained a nearly complete assignment of the side-chain aliphatic C-13 resonances, the side-chain H-1 chemical shifts can be assigned in a semiautomatic fashion using 3D N-15-resolved and C-13-resolved NOESY experiments measured with a randomly partially protonated protein sample. We also discuss perspectives for structure determination of larger proteins by using novel strategies which are based on the {H-1, H-1} NOEs in combination with multiple residual dipolar couplings between adjacent C-13 spins determined with 2D C-13-only experiments.
引用
收藏
页码:167 / 179
页数:13
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