The use of heteronuclear cross-polarization for backbone assignment of 2H-, 15N- and 13C-labeled proteins: A pulse scheme for triple-resonance 4D correlation of sequential amide protons and 15N |
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Authors: | Masahiro Shirakawa Markus Wälchli Masato Shimizu Yoshimasa Kyogoku |
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Affiliation: | (1) Institute for Protein Research, Osaka University, Suita, Osaka 565, Japan;(2) Bruker Japan, 21-5, Ninomiya 3-chome, Tsukuba, Ibaraki 305, Japan |
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Abstract: | Summary A new four-dimensional pulse scheme is described for the main-chain assignment of proteins by means of the J connectivity of the amide proton and nitrogen resonances of adjacent residues. Since the new experiment, 4D CP-HN(COCA)NH, involves heteronuclear cross-polarization for magnetization transfer from 13C=O to 15N via 13C, a relatively strong WALTZ-16 decoupling rf field is applied to 13C during magnetization transfer. Consequently, 13C is effectively decoupled from its attached 2H in the case of deuterated proteins, in the absence of a decoupling rf field for 2H. This efficiently improves the sensitivity of the experiment through 13C line narrowing. The experiment was performed on a randomly 60% deuterated protein, and the sensitivity of the final 4D spectrum was found to be excellent. |
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Keywords: | Heteronuclear NMR Deuterium labeling Cross-polarization Triple-resonance experiment Pulse scheme |
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