Method of using G-matrix Fourier transformation nuclear magnetic resonance (GFT NMR) spectroscopy for rapid chemical shift assignment and secondary structure determination of proteins

A method for sequentially assigning chemical shift values of an α-proton, 1Hα, an α-carbon, 13Cα, a polypeptide backbone carbonyl carbon, 13C′, a polypeptide backbone amide nitrogen, 15N, and a polypeptide backbone amide proton, 1HN, of a protein molecule comprising:

• providing a protein sample;
• conducting a set of G matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiments on the protein sample comprising:
• (1) a (5,2)D [HACACONHN] GFT NMR experiment to measure and connect the chemical shift values of the α-proton of amino acid residue i−1, 1Hαi−1, the α-carbon of amino acid residue i−1, 13Cαi−1, the polypeptide backbone carbonyl carbon of amino acid residue i−1, 13C′i−1, the polypeptide backbone amide nitrogen of amino acid residue i, 15Ni, and the polypeptide backbone amide proton of amino acid residue i, 1HNi and (2) a (5,2)D [HACA,CONHN] GFT NMR experiment to measure and connect the chemical shift values of 1Hαi−1, 13Cαi−1, 13C′i−1, the polypeptide backbone amide nitrogen of amino acid residue i−1, 15Ni−1, and the polypeptide backbone amide proton of amino acid residue i−1, 1HNi−1;
• and
• obtaining sequential assignments of the chemical shift values of 1Hα, 13Cα, 13C′, 15N, and 1HN by (i) matching the chemical shift values of 1Hαi−1, 13Cαi−1, and 13C′i−1 measured by said (5,2)D [HACACONHN] GFT NMR experiment with the chemical shift values of 1Hαi−1, 13Cαi−1, and 13C′i−1 measured by said (5,2)D [HACA,CONHN] GFT NMR experiment, (ii) using the chemical shift values of 1Hαi−1, 13Cαi−1, and 13C′i−1 to identify the type of amino acid residue i−1, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using said chemical shift values to locate secondary structure elements within the polypeptide chain.

A method for sequentially assigning chemical shift values of an α-proton, 1Hα, an α-carbon, 13Cα, a polypeptide backbone carbonyl carbon, 13C′, a polypeptide backbone amide nitrogen, 15N, and a polypeptide backbone amide proton, 1HN, of a protein molecule comprising:

• providing a protein sample;
• conducting a set of G matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiments on the protein sample comprising:
• (1) a (5,3)D [HACACONHN] GFT NMR experiment to measure and connect the chemical shift values of the α-proton of amino acid residue i−1, 1Hαi−1, the α-carbon of amino acid residue i−1, 13Cαi−1, the polypeptide backbone carbonyl carbon of amino acid residue i−1, 13C′i−1, the polypeptide backbone amide nitrogen of amino acid residue i, 15Ni, and the polypeptide backbone amide proton of amino acid residue i, 1HNi and (2) a (5,3)D [HACA,CONHN] GFT NMR experiment to measure and connect the chemical shift values of 1Hαi−1, 13Cαi−1, 13C′i−1, the polypeptide backbone amide nitrogen of amino acid residue i−1, 15Ni−1, and the polypeptide backbone amide proton of amino acid residue i−1, 1HNi−1;
• and
• obtaining sequential assignments of the chemical shift values of 1Hα, 13Cα, 13C′, 15N, and 1HN by (i) matching the chemical shift values of 1Hαi−1, 13Cαi−1, and 13C′i−1 measured by said (5,3)D [HACACONHN] GFT NMR experiment with the chemical shift values of 1Hαi−1 , 13Cαi−1, and 13C′i−l measured by said (5,3)D [HACA,CONHN] GFT NMR experiment, (ii) using the chemical shift values of 1Hαi−1, 13Cαi−1, and 13C′i−1 to identify the type of amino acid residue i−1, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using said chemical shift values to locate secondary structure elements within the polypeptide chain.

A method for sequentially assigning chemical shift values of α- and β-carbons, 13Cα/β, a polypeptide backbone carbonyl carbon, 13C′, a polypeptide backbone amide nitrogen, 15N, and a polypeptide backbone amide proton, 1HN, of a protein molecule comprising:

• providing a protein sample;
• conducting a set of G matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiments on the protein sample comprising:
• (1) a (4,3)D [CBCACONHN] GFT NMR experiment to measure and connect the chemical shift values of the α- and β-carbons of amino acid residue i−1, 13Cα/βi−1, the polypeptide backbone carbonyl carbon of amino acid residue i−1, 13C′i−1, the polypeptide backbone amide nitrogen of amino acid residue i, 15Ni, and the polypeptide backbone amide proton of amino acid residue i, 1HNi and (2) a (4,3)D [CBCA,CONHN] GFT NMR experiment to measure and connect the chemical shift values of 13Cα/βi−1, 13C′i−1, the polypeptide backbone amide nitrogen of amino acid residue i−1, 15Ni−1, and the polypeptide backbone amide proton of amino acid residue i−1, 1HNi−1;
• and
• obtaining sequential assignments of the chemical shift values of 13Cα/β, 13C′, 15N, and 1HN by (i) matching the chemical shift values of 13Cα/βi−1 and 13C′i−1 measured by said (4,3)D [CBCACONHN] GFT NMR experiment with the chemical shift values of 13Cα/βi−1 and 13C′i−1 measured by said (4,3)D [CBCA,CONHN] GFT NMR experiment, (ii) using the chemical shift values of 13Cα/βi−1 and 13C′i−1 to identify the type of amino acid residue i−1, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using said chemical shift values to locate secondary structure elements within the polypeptide chain.

A method for sequentially assigning chemical shift values of α- and β-carbons, 13Cα/β, a polypeptide backbone amide nitrogen, 15N, and a polypeptide backbone amide proton, 1HN, of a protein molecule comprising:

• providing a protein sample;
• conducting a set of G matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiments on the protein sample comprising:
• (1) a (4,3)D [HNNCACBCA] GFT NMR experiment to measure and connect the chemical shift values of the α- and β-carbons of amino acid residue i−1, 13Cα/βi−1, the α-carbon of amino acid residue i−1, 13Cαi−1, the polypeptide backbone amide nitrogen of amino acid residue i−1, 15Ni−1, and the polypeptide backbone amide proton of amino acid residue i−1, 1HNi−1 and (2) a GFT NMR experiment selected from the group consisting of a (4,3)D [HNN(CO)CACBCA] GFT NMR experiment, a (4,3)D [CBCACA(CO)NHN] GFT NMR experiment, and a (5,3)D [HBHACBCACA(CO)NHN] GFT NMR experiment to measure and connect the chemical shift values of 13Cα/βi−1, 13Cαi−1, the polypeptide backbone amide nitrogen of amino acid residue i, 15Ni, and the polypeptide backbone amide proton of amino acid residue i, 1HNi;
• and
• obtaining sequential assignments of the chemical shift values of 13Cα/β, 15N, and 1HN by (i) matching the chemical shift values of 13Cα/βi−1 measured by said GFT NMR experiment selected from the group consisting of a (4,3)D [HNN(CO)CACBCA] GFT NMR experiment, a (4,3)D [CBCACA(CO)NHN] GFT NMR experiment, and a (5,3)D [HBHACBCACA(CO)NHN] GFT NMR experiment with the chemical shift values of 13Cα/βi−1 measured by said (4,3)D [HNNCACBCA] GFT NMR experiment, (ii) using the chemical shift values of 13Cα/βi−1 to identify the type of amino acid residue i−1, and (iii) mapping sets of sequentially connected chemical shift values to the amino acid sequence of the polypeptide chain and using said chemical shift values to locate secondary structure elements within the polypeptide chain.

A method for assigning chemical shift values of γ-, δ-, and ε-aliphatic sidechain protons, 1Hγ/δ/ε, and chemical shift values of γ-, δ-, and ε-aliphatic sidechain carbons located peripheral to β-carbons, 13Cγ/δ/ε, of a protein molecule comprising:

• providing a protein sample;
• conducting a set of G matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiments on the protein sample comprising:
• (1) a (5,3)D [HCC,CH—COSY] GFT NMR experiment to measure and connect the chemical shift values of a proton of amino acid residue i−1, 1Hi−1, a carbon of amino acid residue i−1 coupled to 1Hi−1, 13Ci−1, a carbon coupled to 13Ci−1, 13Ci−1coupled, and a proton coupled to 13Ci−1coupled, 1Hi−1coupled, and (2) a(5,3)D [HBHACBCACA(CO)NHN] GFT NMR experiment to measure and connect the chemical shift values of α- and β-protons of amino acid residue i−1, 1Hα/βi−1, and α- and β-carbons of amino acid residue i−1, 13Cα/βi−1;
• and
• obtaining assignments of the chemical shift values of 1Hγ/δ/ε and 13Cγ/δ/ε by (i) identifying 1Hi−1, 13Ci−1, 13Ci−1coupled, and 1Hi−1coupled measured by said (5,3)D [HCC,CH—COSY] GFT NMR experiment as 1Hαi−1, 13Cαi−1, 13Cβi−1, and 1Hβi−1, respectively, and thereby matching the chemical shift values of 1Hα/βi−1, and 13Cα/βi−1 with the chemical shift values of 1Hα/βi−1 and 13Cα/βi−1 measured by said (5,3)D HBHACBCACA(CO)NHN] GFT NMR experiment, and (ii) using the chemical shift values of 1Hα/βi−1 and 13Cα/βi−1 in conjunction with other chemical shift connections from said (5,3)D [HCC,CH—COSY] GFT NMR experiment to measure the chemical shift values of 1Hγ/δ/εi−1 and 13Cγ/δ/εi−1.

A method for assigning chemical shift values of γ-, δ-, and ε-aliphatic sidechain protons, 1Hγ/δ/ε, and chemical shift values of γ-, δ-, and ε-aliphatic sidechain carbons located peripheral to β-carbons, 13Cγ/δ/ε, of a protein molecule comprising:

• providing a protein sample;
• conducting a set of G matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiments on the protein sample comprising:
• (1) a (4,2)D [HCCH—COSY] GFT NMR experiment to measure and connect the chemical shift values of a proton of amino acid residue i−1, 1Hi−1, a carbon of amino acid residue i−1 coupled to 1Hi−1, 13Ci−1, a carbon coupled to 13Ci−1, 13Ci−1coupled, and a proton coupled to 13Ci−1coupled, 1Hi−1coupled, and (2) a (5,3)D [HBHACBCACA(CO)NHN] GFT NMR experiment to measure and connect the chemical shift values of α- and β-protons of amino acid residue i−1, 1Hα/βi−1, and α- and β-carbons of amino acid residue i−1, 13Cα/βi−1;
• and
• obtaining assignments of the chemical shift values of 1Hγ/δ/ε and 13Cγ/δ/ε by (i) identifying 1Hi−1, 13Ci−1, 13Ci−1coupled, and 1Hi−1coupled measured by said (4,2)D [HCC,H—COSY] GFT NMR experiment as 1Hαi−1, 13Cαi−1, 13Cβi−1, and 1Hβi−1, respectively, and thereby matching the chemical shift values of 1Hα/βi−1 and 13Cα/βi−1 with the chemical shift values of 1Hα/βi−1 and 13Cα/βi−1 measured by said (5,3)D HBHACBCACA(CO)NHN] GFT NMR experiment, and (ii) using the chemical shift values of 1Hα/βi−1 and 13Cα/βi−1 in conjunction with other chemical shift connections from said (4,2)D [HCCH—COSY] GFT NMR experiment to measure the chemical shift values of 1Hγ/δ/εi−1 and 13Cγ/δ/εi−1.

A method for assigning chemical shift values of a γ-carbon, 13Cγ, a δ-carbon, 13Cδ, and a δ-proton, 1Hδ, of an amino acid residue containing an aromatic spin system in a protein molecule comprising:

• providing a protein sample;
• conducting a set of G matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiments on the protein sample comprising:
• (1) a (5,3)D [HBCBCGCDHD] GFT NMR experiment to measure and connect the chemical shift values of a β-proton of amino acid residue i−1, 1Hβi−1, a β-carbon of amino acid residue i−1, 13Cβi−1, a γ-carbon of amino acid residue i−1, 13Cγi−1, a δ-carbon of amino acid residue i−1, 13Cδi−1, and a δ-proton of amino acid residue i−1, 1Hδi−1, and (2) a (5,3)D [HBHACBCACA(CO)NHN] GFT NMR experiment to measure and connect the chemical shift values of 1Hβi−1 and 13Cβi−1;
• and
• obtaining assignments of the chemical shift values of 13Cγ, 13Cδ, and 1Hδ by (i) matching the chemical shift values of 1Hβi−1 and 13Cβi−1 measured by said (5,3)D HBCBCACA(CO)NHN GFT NMR experiment with the chemical shift values of 1Hβi−1 and 13Cβi−1 measured by said (5,3)D [HBCBCGCDHD] GFT NMR experiment, and (ii) using the chemical shift values of 13Cγ, 13Cδ, and 1Hδ to identify the type of amino acid residue containing the aromatic spin system.

A method for assigning chemical shift values of aliphatic and aromatic protons and aliphatic and aromatic carbons of an amino acid residue containing aliphatic and aromatic spin systems in a protein molecule comprising:

• providing a protein sample;
• conducting a set of G matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiments on the protein sample comprising:
• (1) a first GFT NMR experiment, which is selected from the group consisting of a (5,3)D [HCC,CH—COSY] GFT NMR experiment, a (4,2)D [HCCH—COSY] GFT NMR experiment, a (5,2)D [HCCCH—COSY] GFT NMR experiment, and a (5,3)D [HCCCH—COSY] GFT NMR experiment and is acquired for the aliphatic spin system, to measure and connect the chemical shift values of α- and β-protons of amino acid residue i, 1Hα/βi, α- and β-carbons of amino acid residue i, 13Cα/βi, a γ-carbon of amino acid residue i, 13Cγi, and (2) a second GFT NMR experiment, which is selected from the group consisting of a (5,3)D [HCC,CH—COSY] GFT NMR experiment, a (4,2)D [HCCH—COSY] GFT NMR experiment, a (5,2)D [HCCCH—COSY] GFT NMR experiment, and a (5,3)D [HCCCH—COSY] GFT NMR experiment and is acquired for the aromatic spin system, to measure and connect the chemical shift values of 13Cγi and other aromatic protons and carbons of amino acid residue i;
• and
• obtaining assignments of the chemical shift values of the aliphatic and aromatic protons and aliphatic and aromatic carbons by matching the chemical shift value of 13Cγi measured by said first GFT NMR experiment with the chemical shift value of 13Cγi measured by said second GFT NMR experiment.

A method for obtaining assignments of chemical shift values of 1H, 13C, and 15N of a protein molecule comprising:

• providing a protein sample;
• and
• conducting five G matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiments on the protein sample, wherein (1) a first experiment is a (4,3)D [HNNCACBCA] GFT NMR experiment for obtaining intraresidue correlations of chemical shift values;
• (2) a second experiment is a (5,3)D [HBHACBCACA(CO)NHN] GFT NMR experiment for obtaining interresidue correlations of chemical shift values;
• (3) a third experiment is a (5,3)D [HCC,CH—COSY] GFT NMR experiment for obtaining assignments of aliphatic sidechain chemical shift values;
• (4) a fourth experiment is a (5,3)D [HBCBCGCDHD] GFT NMR experiment for linking chemical shift values of aliphatic protons, 1Hβ and 13Cβ, and aromatic protons, 13Cδ and 1Hδ;
• and (5) a fifth experiment is a (4,2)D [HCCH—COSY] GFT NMR experiment for obtaining assignments of aromatic sidechain chemical shift values.