Method of using G-matrix fourier transformation nuclear magnetic resonance (GFT NMR) spectroscopy for rapid chemical shift assignment and secondary structure determination of proteins
View Patent ↗The present invention presents a new approach to rapidly obtaining precise high-dimensional NMR spectral information, named “GFT NMR spectroscopy”, which is based on the phase sensitive joint sampling of the indirect dimensions spanning a subspace of a conventional NMR experiment. The phase-sensitive joint sampling of several indirect dimensions of a high-dimensional NMR experiment leads to largely reduced minimum measurement times when compared to FT NMR. This allows one to avoid the “sampling limited” data collection regime. Concomitantly, the analysis of the resulting chemical shift multiplets, which are edited by the G-matrix transformation, yields increased precision for the measurement of the chemical shifts. Additionally, methods of conducting specific GFT NMR experiments as well as methods of conducting a combination of GFT NMR experiments for rapidly obtaining precise chemical shift assignment and determining the structure of proteins or other molecules are disclosed.
1. A method for assigning chemical shift values of γ-, δ-, and ε-aliphatic sidechain protons, 1 H γ/δ/ε , and chemical shift values of γ-, δ-, and ε-aliphatic sidechain carbons located peripheral to β-carbons, 13 C γ/δ/ε , of a protein molecule comprising:
providing a protein sample;
conducting a set of Fourier transformation (FT) nuclear magnetic resonance (NMR) experiments on the protein sample comprising: (1) a (5,3)D[ HCC ,CH—COSY] FT NMR experiment to measure and connect the chemical shift values of a proton of amino acid residue i−1, 1 H i−1 , a carbon of amino acid residue i−1 coupled to 1 H i−1 , 13 C i−1 , a carbon coupled to 13 C i−1 , 13 C i−1 coupled , and a proton coupled to 13 C i−1 coupled , 1 H i−1 coupled , and (2) a (5,3)D [ HBHACBCACA (CO)NHN] FT NMR experiment to measure and connect the chemical shift values of α- and β-protons of amino acid residue i−1, 1 H α/β i−1 , and α- and β-carbons of amino acid residue i−1, 13 C α/β i−1 ; and
obtaining assignments of the chemical shift values of 1 H γ/δ/ε and 13 C γ/δ/ε by (i) identifying 1 H i−1 , 13 C i−1 , 13 C i−1 coupled , and 1 H i−1 coupled measured by said (5,3)D [ HCC ,CH—COSY] FT NMR experiment as 1 H α i−1 , 13 C α i−1 , 13 C β i−1 , and 1 H β i−1 , respectively, and thereby matching the chemical shift values of 1 H α/β i−1 and 13 C α/β i−1 with the chemical shift values of 1 H α/β i−1 and 13 C α/β i−1 measured by said (5,3)D [ HBHACBCACA (CO)NHN] FT NMR experiment, and (ii) using the chemical shift values of 1 H α/β i−1 and 13 C α/β i−1 in conjunction with other chemical shift connections from said (5,3)D [ HCC ,CH—COSY] FT NMR experiment to measure the chemical shift values of 1 H γ/δ/ε i−1 and 13 C γ/δ/ε i−1 .
2. The method according to claim 1 further comprising:
subjecting the protein sample to nuclear Overhauser enhancement spectroscopy (NOESY) to deduce the tertiary structure of the protein molecule.
3. The method according to claim 1 further comprising:
subjecting the protein sample to NMR experiments that measure scalar coupling constants to deduce the tertiary structure of the protein molecule.
4. The method according to claim 1 further comprising:
subjecting the protein sample to NMR experiments that measure residual dipolar coupling constants to deduce the tertiary structure of the protein molecule.
5. A method for assigning chemical shift values of γ-, δ-, and ε-aliphatic sidechain protons, 1 H γ/δ/ε , and chemical shift values of γ-, δ-, and ε-aliphatic sidechain carbons located peripheral to β-carbons, 13 C γ/δ/ε , of a protein molecule comprising:
providing a protein sample;
conducting a set of Fourier transformation (FT) nuclear magnetic resonance (NMR) experiments on the protein sample comprising: (1) a (4,2)D [ HCC H—COSY] FT NMR experiment to measure and connect the chemical shift values of a proton of amino acid residue i−1, 1 H i−1 , a carbon of amino acid residue i−1 coupled to 1 H i−1 , 13 C i−1 , a carbon coupled to 13 C i−1 , 13 C i−1 coupled , and a proton coupled to 13 C i−1 coupled , 1 H i−1 coupled ,and (2) a (5,3)D [ HBHACBCACA (CO)NHN] FT NMR experiment to measure and connect the chemical shift values of α- and β-protons of amino acid residue i−1, 1 H α/β i−1 , and α- and β-carbons of amino acid residue i−1, 13 C α/β i−1 ; and
obtaining assignments of the chemical shift values of 1 H γ/δ/ε and 13 C γ/δ/ε by (i) identifying 1 H i−1 , 13 C i−1 , 13 C i−1 coupled , and 1 H i−1 coupled measured by said (4,2)D [ HCC H—COSY] FT NMR experiment as 1 H α i−1 , 13 C α i−1 , 13 C β i−1 , and 1 H β i−1 , respectively, and thereby matching the chemical shift values of 1 H α/β i−1 and 13 C α/β i−1 with the chemical shift values of 1 H α/β i−1 and 13 C α/β i−1 measured by said (5,3)D HBHACBCACA (CO)NHN] FT NMR experiment, and (ii) using the chemical shift values of 1 H α/β i−1 and 13 C α/β i−1 in conjunction with other chemical shift connections from said (4,2)D [ HCC H—COSY] FT NMR experiment to measure the chemical shift values of 1 H γ/δ/ε i−1 and 13 C γ/δ/ε i−1 .
6. The method according to claim 5 further comprising:
subjecting the protein sample to nuclear Overhauser enhancement spectroscopy (NOESY) to deduce the tertiary structure of the protein molecule.
7. The method according to claim 5 further comprising:
subjecting the protein sample to NMR experiments that measure scalar coupling constants to deduce the tertiary structure of the protein molecule.
8. The method according to claim 5 further comprising:
subjecting the protein sample to NMR experiments that measure residual dipolar coupling constants to deduce the tertiary structure of the protein molecule.