New NMR experiments

   Our research work on NMR methodology focuses on the development of new experiments for fast acquiring NMR data, which are used to determine the 3D (three-dimensional) structures of proteins, DNA, RNA and their complexes. Such structures are the foundation for rational structure-based drug design and are essential to the continuing development of new medicines and understanding of human disease.

1. Time-Sharing NOESY experiments

   This work was published on Journal of Biomolecular NMR 27 (3): 193-203 (2003).   

   [pulse sequences and parameter files on 800 MHz instrument, detailed description]

    

2. GFT-NMR experiments

   This work was published on Journal of Biomolecular NMR 29 (4): 467-476 (2004).   

   [pulse sequences and parameter files on 800 MHz instrument, detailed description]

    

3. IP-COSY, a Totally In-Phase and Sensitive COSY

   This work was published on Magnetic Resonance in Chemistry 43, 372-379 (2005).  

   [pulse sequences and parameter files on 600 MHz instrument, detailed description]

  Detailed description:

    1) ¹HC and ¹HN Total NOE Correlations in a Single 3D NMR Experiment.

          ¹⁵N and ¹³C Time-Sharing in t1 and t2 Dimensions for Simultaneous Data Acquisition

   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.

   (A) 2D CN-H HSQC pulse sequence

   (B) Comparison of water suppression effect with the 2D CN-H HSQC pulse sequence

   (C) Comparisons of sensitivities of the 2D CN-H HSQC and other version

(a) 2D CN-H HSQC (this work); (b) 2D CN-H HSQC (Griesinger)

   (D) 3D 2TS CN-CN-H NOESY pulse sequence

   (E) 3D TS H-CN-H NOESY pulse sequence

   (F) Unambiguous assignments of NOE cross peaks

   (G) Conclusion

   The proposed novel 3D CN-CN-H HSQC-NOESY-HSQC employed a 2TS strategy, thereby experimental time was reduced by 75% compared with conventional experiment;

1. Four types of NOE connections all were included in the 3D CN-CN-H spectra;
2. 2D CN-H HSQC and 3D H-CN-H experiments were improved and optimized. So the three experiments promised good water suppression and strong signals;
3. The digital resolutions of 13C and 15N were optimized independently;
4. Combining 3D CN-CN-H and H-CN-H NOESY experiments, NOE assignment ambiguity was significantly reduced.

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    2) (3,2)D GFT-NMR Experiments for Fast Collection of Protein Data

   High throughput structure determination of proteins will contribute to the success of proteomics investigations. The G-Matrix Fourier Transformation NMR (GFT-NMR) method significantly shortens experimental time by reducing the number of the dimensions of data acquisition for isotopically labeled proteins (Kim, S. and Szyperski, T. (2003) GFT NMR, a new approach to rapidly obtain precise high-demensional NMR spectral information, J. Am. Chem. Soc. 125, 1385). We demonstrate herein a suite of ten 2D or (3,2)D GFT-NMR experiments using 13C/15N-labeled ubiquitin. These experiments were completed within 18 hours, representing a 4- to 18-fold reduction in data acquisition time compared to the corresponding conventional 3D experiments. A subset of the GFT-NMR experiments, (3,2)D HNCO, HNCACB, HN(CO)CACB, and 2D 1H-15N HSQC, which are necessary for backbone assignments, were carried out within 6 hours. To facilitate the analysis of the GFT-NMR spectra, we developed automated procedures for viewing and analyzing the GFT-NMR spectra. Our overall strategy allows (3,2)D GFT-NMR experiments to be readily performed and analyzed. Nevertheless, the increase in spectral overlap and the reduction in signal sensitivity in these fast NMR experiments presently limit their application to relatively small proteins.

   (A) (3,2)D HNCO pulse sequence

   (B) (3, 2)D HN(CO)CACB spectra

   (C) Automated matching central peak and doublet

   (D) Comparison of experimental times and sensitivities

   (E) Conclusion

1. This suite of ten experiments can be completed in about 18 hours, while the necessary experiments for backbone assignments, (3, 2)D HNCACB, HN(CO)CACB, HNCO, and 2D 15N-1H HSQC can be carried out in less than 6 hours.
2. A scheme (gft_match.awk) for automatic analysis GFT-NMR data was established.
3. The NMR data analysis software, SPARKY, is extended to analyze GFT-NMR data.

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    3) IP-COSY, a Totally In-Phase and Sensitive COSY

   The IP-COSY experiment presented in this work gives an in-phase spectral presentation in both F1 and F2 dimensions by a combined use of a constant evolution time (CT) in t1 and a symmetrical refocusing period before t2. Compared to DQF-COSY and CT-COSY, IP-COSY further alleviates the effect of signal reduction due to small ratio p (=J/linewidth), showing: (1) improved line shape and cross-peak definition, and (2) especially, enhancement in signals of the peaks of small active J coupling constant and the peaks of broader linewidth. A new strategy was adopted to effectively eliminate or reduce artifactual peaks by adding 0.1-0.2 ms variation to the time delays of the CT period used for each scan of the FID in IP-COSY and CT-COSY. 3JHH coupling constants of larger than 4 Hz in the fingerprint region of peptides can be directly derived from the separation of doublets. IP-COSY cross-peaks are stronger than DQF-COSY by 4- to 20-fold for tested peptides and oligonucleotides (Mw < 8 kDa) with acquisition and processing parameters used in the work, and they are easier to identify than those in CT-COSY. The overall improvement in IP-COSY should make the detection/autodetection of the COSY cross-peaks and the measurements of the various coupling constants more easily achieved, providing valuable information for the structure elucidation of peptides/small proteins and oligonucleotides. 

   (A) Pulse sequences of IP-COSY

   (B) Comparisons of DQF-COSY, CT-COSY and IP-COSY of two peptides

   (C) Four-bond correlations from IP-COSY

   (D) Comparisons of DQF-COSY, CT-COSY and IP-COSY of a DNA sample

   (E) Conclusion

1. Magnetization in t1 and t2 dimensions is inphase;
2. Artifacts from Strong couplings are removed;
3. 2J and 3J can be measured directly; 4J connections can be obtained;
4. The sensitivity of IP-COSY is about 2 to 3-time of that of DQF-COSY for small peptides (<50 amino acids). And for medium sized peptides (such as lysozyme), sensitivity of IP-COSY is about 5 to 10-time of that of DQF-COSY.

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