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Spectroscopy
Volume 16, Issue 1, Pages 1-13
http://dx.doi.org/10.1155/2002/828353

Protein Dynamics Measurements by 3D HNCO Based NMR Experiments

Youlin Xia,1 Kong Hung Sze,1 Ning Li,2 Pang Chui Shaw,3 and Guang Zhu1

1Department of Biochemistry, The Hong Kong University of Science and Technology, Kowloon, Hong Kong, China
2Department of Biology, The Hong Kong University of Science and Technology, Kowloon, Hong Kong, China
3Department of Biochemistry, The Chinese University of Hong Kong, Shatin, Hong Kong, China

Copyright © 2002 Hindawi Publishing Corporation. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Protein dynamics can be characterized by relaxation parameters obtained from traditional 2D HSQC based NMR experiments. This approach is hampered when applied to proteins with severe spectral overlap. In the present work, several novel 3D TROSY-HNCO and 3D HSQC-HNCO based NMR experiments were applied for measuring 15N T1, T2 and 1H-15N NOE with improved spectral dispersion by introducing a third 13C dimension. The number of phase cycling steps in these 3D pulse sequences was restricted to two in order to minimize the time required to perform the dynamics measurements. For a uniformly 100% 15N, 100% 13C, and 70%2H-labelled trichosanthin sample (~27 kDa, 1.0 mM) at 30°C, the sensitivity of 3D TROSY-HNCO based experiment was, on the average, enhanced by 72% compared to that of 3D HSQC-HNCO based experiments. However, the 3D HSQC-HNCO based experiments should be more effective for non-deuterated proteins with smaller molecular weights and seriously overlapped 2D HSQC spectra. Results from the 3D TROSY-HNCO and 3D HSQC-HNCO based experiments were in good agreement with those obtained from traditional 2D HSQC based experiments.