Authors
Yingqi Xu, Yu Zheng, and Daiwen Yang
Summary
NMR spectroscopy has been widely used to determine high resolution structures of proteins and protein complexes in solution1, 2. However, it is is not suitable for larger proteins (> 30 kDa) due to increased signal decay rates. Deuterium labeling of protein samples overcomes this problem3, but deuteration makes sample preparation much harder and structure determination more difficult4, 5. Here we introduce a strategy that uses non-deuterated samples6. Backbone and side chain resonances of large proteins are assigned from 3D TROSY-HNCA7, 3D MQ-CCH-TOCSY8 and 4D 15N,13C-edited NOESY9 spectra. The three spectra with reasonable quality can be obtained within ~11 days using a uniformly 13C,15N-labeled sample at a protein concentration of ~1 mM. Unlike the conventional strategy mainly relying on through-bond correlation experiments, the strategy used here mainly uses a through-space correlation experiment (4D 15N,13C-edited NOESY) to obtain intra-residue and sequential correlations, which are separated from other inter-residue NOE correlations using the 3D HNCA and CCH-TOCSY experiments. Similar to the conventional strategy, sequential assignment is achieved from matching intra-residue and sequential correlations. Using this strategy, one can obtain nearly complete backbone assignments and >80% side chain assignments for monomeric proteins < 42 kDa and multimeric proteins < 65 kDa.Further details
The protocol was published on Protocol Exchange in 2006. To see the entire protocol, click on the source link.Advertisement
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