(3,2)D GFT-NMR experiments for fast data collection from proteins.
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) J. Am. Chem. Soc. 125, 1385). We demonstrate herein a suite of ten 3D-->2D or (3,2)D GFT-NMR experiments using (13)C/(15)N-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 (1)H-(15)N 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.
Xia
Youlin
Y
Department of Chemistry, University of Houston, Houston, TX 77004-5003, U.S.A.
Zhu
Guang
G
Veeraraghavan
Sudha
S
Gao
Xiaolian
X
D002247:Carbon Isotopes; D003627:Data Interpretation, Statistical; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes; D011506:Proteins / Q000737:chemistry*; D025801:Ubiquitin / Q000737:chemistry
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D025801:Ubiquitin