Simultaneous data acquisition in time-sharing (TS) multi-dimensional NMR experiments has been shown an effective means to reduce experimental time, and thus to accelerate structure determination of proteins. This has been accomplished by spin evolution time-sharing of the X and Y heteronuclei, such as 15N and 13C, in one of the time dimensions. In this work, we report a new 3D TS experiment, which allows simultaneous 13C and 15N spin labeling coherence in both t1 and t2 dimensions to give four NOESY spectra in a single 3D experiment. These spectra represent total NOE correlations between 1HN and 1HC resonances. This strategy of double time-sharing (2TS) results in an overall four-fold reduction in experimental time compared with its conventional counterpart. This 3D 2TS CN-CN-H HSQC-NOESY-HSQC pulse sequence also demonstrates improvements in water suppression, 15N spectral resolution and sensitivity, which were developed based on 2D TS CN-H HSQC and 3D TS H-CN-H NOESY-HSQC experiments. Combining the 3D TS and the 3D 2TS NOESY experiments, NOE assignment ambiguities and errors are considerably reduced. These results will be useful for rapid protein structure determination to complement the effort of discerning the functions of diverse genomic proteins.
Bibliographical noteFunding Information:
This work is supported by grants from NIH to Xiaolian Gao (GM49957) and by a grant to the Northeast Structural Genomics Consortium from the Protein Structure Initiative of the NIH (P50 GM62413), and the Ontario Research and Development Challenge Fund to Cheryl H. Arrowsmith. We thank Drs Karen L. Maxwell and Glen Legge for useful discussions and helpful comments. The pulse sequences and AU programs are available at http://www.chem.uh.edu/Faculty/Gao/ResearchWeb-pages/index.html.
- Protein NMR
- Simultaneous data acquisition