Method of using G-matrix Fourier transformation nuclear magnetic resonance (GFT NMR) spectroscopy for rapid chemical shift assignment and secondary structure determination of proteins
DCFirst Claim
1. A method of conducting a (N,N−
- K) dimensional (D) G-matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiment, wherein N is the dimensionality of an N-dimensional (ND) Fourier transformation (FT) NMR experiment and K is the desired reduction in dimensionality relative to N, said method comprising;
providing a sample;
applying radiofrequency pulses for the ND FT NMR experiment to the sample;
selecting m indirect chemical shift evolution periods of the ND FT NMR experiment, wherein m equals K+1;
jointly sampling the m indirect chemical shift evolution periods;
independently cosine and sine modulating NMR signals detected in a direct dimension to generate (N−
K)D basic NMR spectra comprising frequency domain signals with 2K chemical shift multiplet components, thereby enabling phase-sensitive sampling of all jointly sampled m indirect chemical shift evolution periods; and
transforming the (N−
K) D basic NMR spectra into (N−
K) D phase-sensitively edited basic NMR spectra, wherein the 2K chemical shift multiplet components of the (N−
K) D basic NMR spectra are edited to yield (N−
K) D phase-sensitively edited basic NMR spectra having individual chemical shift multiplet components.
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Abstract
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.
24 Citations
56 Claims
-
1. A method of conducting a (N,N−
- K) dimensional (D) G-matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiment, wherein N is the dimensionality of an N-dimensional (ND) Fourier transformation (FT) NMR experiment and K is the desired reduction in dimensionality relative to N, said method comprising;
providing a sample;
applying radiofrequency pulses for the ND FT NMR experiment to the sample;
selecting m indirect chemical shift evolution periods of the ND FT NMR experiment, wherein m equals K+1;
jointly sampling the m indirect chemical shift evolution periods;
independently cosine and sine modulating NMR signals detected in a direct dimension to generate (N−
K)D basic NMR spectra comprising frequency domain signals with 2K chemical shift multiplet components, thereby enabling phase-sensitive sampling of all jointly sampled m indirect chemical shift evolution periods; and
transforming the (N−
K) D basic NMR spectra into (N−
K) D phase-sensitively edited basic NMR spectra, wherein the 2K chemical shift multiplet components of the (N−
K) D basic NMR spectra are edited to yield (N−
K) D phase-sensitively edited basic NMR spectra having individual chemical shift multiplet components.- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56)
wherein i=√
{square root over (−
1)}, under conditions effective to edit the chemical shift multiplet components in a time domain.
- K) dimensional (D) G-matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiment, wherein N is the dimensionality of an N-dimensional (ND) Fourier transformation (FT) NMR experiment and K is the desired reduction in dimensionality relative to N, said method comprising;
-
3. The method according to claim 1, wherein said transforming is carried out by applying a F-matrix defined as {circumflex over (F)}(K)={circumflex over (F)}(K−
- 1){circle around (x)}{circumflex over (F)}(1), wherein
under conditions effective to edit the chemical shift multiplet components in a frequency domain.
- 1){circle around (x)}{circumflex over (F)}(1), wherein
-
4. The method according to claim 1 further comprising:
-
selecting m′
indirect chemical shift evolution periods of the (N−
K)D FT NMR experiment, wherein m′
equals K′
+1;
jointly sampling the m′
indirect chemical shift evolution periods;
independently cosine and sine modulating NMR signals detected in a direct dimension to generate (N−
K−
K′
)D basic NMR spectra comprising frequency domain signals with 2K′
chemical shift multiplet components, thereby enabling phase-sensitive sampling of all jointly sampled m′
indirect chemical shift evolution periods; and
transforming the (N−
K−
K′
) D basic NMR spectra into (N−
K−
K′
) D phase-sensitively edited basic NMR spectra, wherein the 2K′
chemical shift multiplet components of the (N−
K−
K′
) D basic NMR spectra are edited to yield (N−
K−
K′
) D phase-sensitively edited basic NMR spectra having individual chemical shift multiplet components.
-
-
5. The method according to claim 4 further comprising:
repeating one or more times said selecting, said jointly sampling, said independently cosine and sine modulating, and said transforming, wherein m′
is modified for each repetition.
-
6. The method according to claim 1 further comprising:
repeating one or more times said selecting, said jointly sampling, said independently cosine and sine modulating, and said transforming, wherein, for each repetition, said selecting comprises selecting m-j indirect chemical shift evolution periods out of the m indirect chemical shift evolution periods, wherein j ranges from 1 to K, under conditions effective to generate 2K-j jth order central peak NMR spectra.
-
7. The method according to claim 1, wherein said applying radiofrequency pulses is carried out by applying radiofrequency pulses of N-dimensional nuclear Overhauser enhancement spectroscopy (NOESY).
-
8. The method according to claim 1, wherein said applying radiofrequency pulses is carried out by applying radiofrequency pulses of N-dimensional transverse relaxation optimized spectroscopy (TROSY).
-
9. The method according to claim 1, wherein said applying radiofrequency pulses is carried out so that spin-spin couplings are measured.
-
10. The method according to claim 9, wherein said spin-spin couplings are residual dipolar spin-spin coupling constants.
-
11. The method according to claim 1, wherein said jointly sampling the m indirect chemical shift evolution periods is achieved with a single continuous acquisition.
-
12. The method according to claim 1, wherein said applying radiofrequency pulses is carried out so that nuclear spin relaxation times are measured by sampling nuclear spin relaxation delays.
-
13. The method according to claim 12 further comprising:
jointly sampling said spin relaxation delays with chemical shift evolution periods.
-
14. The method according to claim 1, wherein N equals 5 and K equals 3 to conduct a (5,2)D [HACACONHN] GFT NMR experiment, wherein (a) said sample is a protein molecule having two consecutive amino acid residues, i−
- 1 and i, and the chemical shift values for the following nuclei are measured;
(1) an α
-proton of amino acid residue i−
1, 1Hα
i−
1;
(2) an α
-carbon of amino acid residue i−
1, 13Cα
i−
1;
(3) a polypeptide backbone carbonyl carbon of amino acid residue i−
1, 13C′
i−
1;
(4) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(5) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 4 chemical shift evolution periods of the 5D FT NMR experiment, 1Hα
i−
1, 13Cα
i−
1, 13C′
i−
1, and 15Ni, and (c) said jointly sampling comprises jointly sampling the 4 chemical shift evolution periods in an indirect time domain dimension, t1(1Hα
i−
1, 13Cα
i−
1, 13C′
i−
1, 15Ni).
- 1 and i, and the chemical shift values for the following nuclei are measured;
-
15. The method according to claim 14, wherein said applying radiofrequency pulses comprises applying radiofrequency pulses for a 5D FT NMR experiment according to the scheme shown in FIG. 6.
-
16. The method according to claim 1, wherein N equals 5 and K equals 3 to conduct a (5,2)D [HACA,CONHN] GFT NMR experiment, wherein (a) said sample is a protein molecule having an amino acid residue, i, and the chemical shift values for the following nuclei are measured:
- (1) an α
-proton of amino acid residue i−
1, 1Hα
i−
1;
(2) an α
-carbon of amino acid residue i−
1, 13Cα
i−
1;
(3) a polypeptide backbone carbonyl carbon of amino acid residue i−
1, 13C′
i−
1;
(4) a polypeptide backbone amide nitrogen of amino acid residue i−
1, 15Ni−
1; and
(5) a polypeptide backbone amide proton of amino acid residue i−
1, 1HNi−
1, (b) said selecting comprises selecting 4 chemical shift evolution periods of the 5D FT NMR experiment, 1Hα
i−
1, 13Cα
i−
1, 13C′
i−
1, and 15Ni−
1, and (c) said jointly sampling comprises jointly sampling the 4 chemical shift evolution periods in an indirect time domain dimension, t1(1Hα
i−
1, 13Cα
i−
1, 13C′
i−
1, 15Ni−
1).
- (1) an α
-
17. The method according to claim 16, wherein said applying radiofrequency pulses comprises applying radiofrequency pulses for a 5D FT NMR experiment according to the scheme shown in FIG. 7A.
-
18. The method according to claim 1, wherein N equals 5 and K equals 2 to conduct a (5,3)D [HACACONHN] GFT NMR experiment, wherein (a) said sample is a protein molecule having two consecutive amino acid residues, i−
- 1 and i, and the chemical shift values for the following nuclei are measured;
(1) an α
-proton of amino acid residue i−
1, 1Hα
i−
1;
(2) an α
-carbon of amino acid residue i−
1, 13Cα
i−
1;
(3) a polypeptide backbone carbonyl carbon of amino acid residue i−
1, 13C′
i−
1;
(4) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(5) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 5D FT NMR experiment, 1Hα
i−
1, 13Cα
i−
1, and 13C′
i−
1, and (c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(1Hα
i−
1, 13Cα
i−
1, 13C′
i−
1).
- 1 and i, and the chemical shift values for the following nuclei are measured;
-
19. The method according to claim 1, wherein N equals 5 and K equals 2 to conduct a (5,3)D [HACA,CONHN] GFT NMR experiment, wherein (a) said sample is a protein molecule having an amino acid residue, i−
- 1, and the chemical shift values for the following nuclei are measured;
(1) an α
-proton of amino acid residue i−
1, 1Hα
i−
1;
(2) an α
-carbon of amino acid residue i−
1, 13Cα
i−
1;
(3) a polypeptide backbone carbonyl carbon of amino acid residue i−
1, 13C′
i−
1;
(4) a polypeptide backbone amide nitrogen of amino acid residue i−
1, 15Ni−
1; and
(5) a polypeptide backbone amide proton of amino acid residue i−
1, 1HNi−
1, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 5D FT NMR experiment, 1Hα
i−
1, 13Cα
i−
1, and 13C′
i−
1, and (c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(1Hα
i−
1, 13Cα
i−
1, 13C′
i−
1).
- 1, and the chemical shift values for the following nuclei are measured;
-
20. The method according to claim 1, wherein N equals 4 and K equals 1 to conduct a (4,3)D [CBCACONHN] GFT NMR experiment, wherein (a) said sample is a protein molecule having two consecutive amino acid residues, i−
- 1 and i, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
-carbons of amino acid residue i−
1, 13Cα
/β
i−
1;
(2) a polypeptide backbone carbonyl carbon of amino acid residue i−
1, 13C′
i−
1;
(3) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(4) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 2 chemical shift evolution periods of the 4D FT NMR experiment, 13Cα
/β
i−
1 and 13C′
i−
1, and (c) said jointly sampling comprises jointly sampling the 2 chemical shift evolution periods in an indirect time domain dimension, t1(13Cα
/β
i−
1, 13C′
i−
1).
- 1 and i, and the chemical shift values for the following nuclei are measured;
-
21. The method according to claim 20, wherein said applying radiofrequency pulses comprises applying radiofrequency pulses for a 4D FT NMR experiment according to the scheme shown in FIG. 8.
-
22. The method according to claim 1, wherein N equals 4 and K equals 1 to conduct a (4,3)D [CBCA,CONHN] GFT NMR experiment, wherein (a) said sample is a protein molecule having an amino acid residue, i−
- 1, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
-carbons of amino acid residue i−
1, 13Cα
/β
i−
1;
(2) a polypeptide backbone carbonyl carbon of amino acid residue i−
1, 13C′
i−
1;
(3) a polypeptide backbone amide nitrogen of amino acid residue i−
1, 15Ni−
1; and
(4) a polypeptide backbone amide proton of amino acid residue i−
1, 1HNi−
1, (b) said selecting comprises selecting 2 chemical shift evolution periods of the 4D FT NMR experiment, 13Cα
/β
i−
1 and 13C′
i−
1, and (c) said jointly sampling comprises jointly sampling the 2 chemical shift evolution periods in an indirect time domain dimension, t1(13Cα
/β
i−
1, 13C′
i−
1).
- 1, and the chemical shift values for the following nuclei are measured;
-
23. The method according to claim 22, wherein said applying radiofrequency pulses comprises applying radiofrequency pulses for a 4D FT NMR experiment according to the scheme shown in FIG. 7B.
-
24. The method according to claim 1, wherein N equals 4 and K equals 1 to conduct a (4,3)D [HNNCACBCA] GFT NMR experiment, wherein (a) said sample is a protein molecule having two consecutive amino acid residues, i−
- 1 and i, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
-carbons of amino acid residues i and i−
1, 13Cα
/β
i/i−
1;
(2) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(3) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 2 chemical shift evolution periods of the 4D FT NMR experiment, 13Cα
/β
i/i−
1 and 13Cα
i/i−
1, and (c) said jointly sampling comprises jointly sampling the 2 chemical shift evolution periods in an indirect time domain dimension, t1(13Cα
/β
i/i−
1, 13Cα
i/i−
1).
- 1 and i, and the chemical shift values for the following nuclei are measured;
-
25. The method according to claim 24, wherein said applying radiofrequency pulses comprises applying radiofrequency pulses for a 4D FT NMR experiment according to the scheme shown in FIG. 9.
-
26. The method according to claim 1, wherein N equals 4 and K equals 2 to conduct a (4,2)D [HNNCACBCA] GFT NMR experiment, wherein (a) said sample is a protein molecule having two consecutive amino acid residues, i−
- 1 and i, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
-carbons of amino acid residues i and i−
1, 13Cα
/β
i/i−
1;
(2) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(3) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 4D FT NMR experiment, 13Cα
/β
i/i−
1, 13Cα
i/i−
1, and 15Ni, and (c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(13Cα
/β
i/i−
1, 13Cα
i/i−
1, 15Ni).
- 1 and i, and the chemical shift values for the following nuclei are measured;
-
27. The method according to claim 1, wherein N equals 4 and K equals 1 to conduct a (4,3)D [HNN(CO)CACBCA] GFT NMR experiment, wherein (a) said sample is a protein molecule having two consecutive amino acid residues, i−
- 1 and i, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
-carbons of amino acid residue i−
1, 13Cα
/β
i−
1;
(2) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(3) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 2 chemical shift evolution periods of the 4D FT NMR experiment, 13Cα
/β
i−
1 and 13Cα
i−
1, and (c) said jointly sampling comprises jointly sampling the 2 chemical shift evolution periods in an indirect time domain dimension, t1(13Cα
/β
i−
1, 13Cα
i−
1).
- 1 and i, and the chemical shift values for the following nuclei are measured;
-
28. The method according to claim 27, wherein said applying radiofrequency pulses comprises applying radiofrequency pulses for a 4D FT NMR experiment according to the scheme shown in FIG. 10.
-
29. The method according to claim 1, wherein N equals 4 and K equals 2 to conduct a (4,2)D [HNN(CO)CACBCA] GFT NMR experiment, wherein (a) said sample is a protein molecule having two consecutive amino acid residues, i−
- 1 and i, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
-carbons of amino acid residue i−
1, 13Cα
/β
i−
1;
(2) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(3) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 4D FT NMR experiment, 13Cα
/β
i−
1, 13Cα
i−
1, and 15Ni; and
(c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(13Cα
/β
i−
1, 13Cα
i−
1, 15Ni).
- 1 and i, and the chemical shift values for the following nuclei are measured;
-
30. The method according to claim 1, wherein N equals 5 and K equals 2 to conduct a (5,3)D [HNNCOCACBCA] GFT NMR experiment, wherein (a) said sample is a protein molecule having two consecutive amino acid residues, i−
- 1 and i, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
-carbons of amino acid residue i−
1, 13Cα
/β
i−
1;
(2) a polypeptide backbone carbonyl carbon of amino acid residue i−
1, 13C′
i−
1, (3) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(4) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 5D FT NMR experiment, 13Cα
/β
i−
1, 13Cα
i−
1, and 13C′
i−
1, (c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(13Cα
/β
i−
1, 13Cα
i−
1, 13C′
i−
1).
- 1 and i, and the chemical shift values for the following nuclei are measured;
-
31. The method according to claim 1, wherein N equals 5 and K equals 3 to conduct a (5,2)D [HNNCOCACBCA] GFT NMR experiment, wherein (a) said sample is a protein molecule having two consecutive amino acid residues, i−
- 1 and i, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
-carbons of amino acid residue i−
1, 13Cα
/β
i−
1;
(2) a polypeptide backbone carbonyl carbon of amino acid residue i−
1, 13C′
−
1, (3) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(4) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 4 chemical shift evolution periods of the 5D FT NMR experiment, 13Cα
/β
i−
1, 13Cα
i−
1, 13C′
i−
1, and 15Ni; and
(c) said jointly sampling comprises jointly sampling the 4 chemical shift evolution periods in an indirect time domain dimension, t1(13Cα
/β
i−
1, 13Cα
i−
1, 13C′
i−
1, 15Ni).
- 1 and i, and the chemical shift values for the following nuclei are measured;
-
32. The method according to claim 1, wherein N equals 4 and K equals 1 to conduct a (4,3)D [CBCACA(CO)NHN] GFT NMR experiment, wherein (a) said sample is a protein molecule having two consecutive amino acid residues, i−
- 1 and i, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
-carbons of amino acid residue i−
1, 13Cα
/β
i−
1;
(2) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(3) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 2 chemical shift evolution periods of the 4D FT NMR experiment, 13Cα
/β
i−
1 and 13Cα
i−
1, and (c) said jointly sampling comprises jointly sampling the 2 chemical shift evolution periods in an indirect time domain dimension, t1(13Cα
/β
i−
1, 13Cα
i−
1).
- 1 and i, and the chemical shift values for the following nuclei are measured;
-
33. The method according to claim 32, wherein said applying radiofrequency pulses comprises applying radiofrequency pulses for a 4D FT NMR experiment according to the scheme shown in FIG. 11.
-
34. The method according to claim 1, wherein N equals 4 and K equals 2 to conduct a (4,2)D [CBCACA(CO)NHN] GFT NMR experiment, wherein (a) said sample is a protein molecule having two consecutive amino acid residues, i−
- 1 and i, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
-carbons of amino acid residue i−
1, 13Cα
/β
i−
1;
(2) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(3) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 4D FT NMR experiment, 13Cα
/β
i−
1, 13Cα
i−
1, and 15Ni, and (c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(13Cα
/β
i−
1, 13Cα
i−
1, 15Ni).
- 1 and i, and the chemical shift values for the following nuclei are measured;
-
35. The method according to claim 1, wherein N equals 5 and K equals 2 to conduct a (5,3)D [CBCACACONHN] GFT NMR experiment, wherein (a) said sample is a protein molecule having two consecutive amino acid residues, i−
- 1 and i, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
-carbons of amino acid residue i−
1, 13Cα
/β
i−
1;
(2) a polypeptide backbone carbonyl carbon of amino acid residue i−
1, 13C′
i−
1, (3) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(4) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 5D FT NMR experiment, 13Cα
/β
i−
1, 13Cα
i−
1, and 13C′
i−
1, (c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(13Cα
/β
i−
1, 13Cα
i−
1, 13C′
i−
1).
- 1 and i, and the chemical shift values for the following nuclei are measured;
-
36. The method according to claim 1, wherein N equals 5 and K equals 3 to conduct a (5,2)D [CBCACACONHN] GFT NMR experiment, wherein (a) said sample is a protein molecule having two consecutive amino acid residues, i−
- 1 and i, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
-carbons of amino acid residue i−
1, 13Cα
/β
i−
1;
(2) a polypeptide backbone carbonyl carbon of amino acid residue i−
1, 13C′
i−
1, (3) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(4) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 4 chemical shift evolution periods of the 5D FT NMR experiment, 13Cα
/β
i−
1, 13Cα
i−
1, 13C′
i−
1, and 15Ni;
(c) said jointly sampling comprises jointly sampling the 4 chemical shift evolution periods in an indirect time domain dimension, t1(13Cα
/β
i−
1, 13Cα
i−
1, 13C′
i−
1, 15Ni).
- 1 and i, and the chemical shift values for the following nuclei are measured;
-
37. The method according to claim 1, wherein N equals 5 and K equals 2 to conduct a (5,3)D [HBHACBCACA(CO)NHN] GFT NMR experiment, wherein (a) said sample is a protein molecule having two amino acid residues, i and i−
- 1, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
-protons of amino acid residue i−
1, 1Hα
/β
i−
1;
(2) α
- and β
-carbons of amino acid residue i−
1, 13Cα
/β
i−
1;
(3) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(4) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 5D FT NMR experiment, 1Hα
/β
i−
1, 13Cα
/β
i−
1, and 13Cα
i−
1, and (c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(1Hα
/β
i−
1, 13Cα
/β
i−
1, 13Cα
i−
1).
- 1, and the chemical shift values for the following nuclei are measured;
-
38. The method according to claim 37, wherein said applying radiofrequency pulses comprises applying radiofrequency pulses for a 5D FT NMR experiment according to the scheme shown in FIG. 12.
-
39. The method according to claim 1, wherein N equals 6 and K equals 3 to conduct a (6,3)D [HBHACBCACACONHN] GFT NMR experiment, wherein (a) said sample is a protein molecule having two amino acid residues, i and i−
- 1, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
protons of amino acid residue i−
1, 1Hα
/β
i−
1;
(2) α
- and β
-carbons of amino acid residue i−
1, 13Cα
/β
i−
1;
(3) a polypeptide backbone carbonyl carbon of amino acid residue i−
1, 13C′
i−
i;
(4) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(5) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 4 chemical shift evolution periods of the 6D FT NMR experiment, 1Hα
/β
i−
1, 13Cα
/β
i−
1, 13Cα
i−
1, and 13C′
i−
1, and (c) said jointly sampling comprises jointly sampling the 4 chemical shift evolution periods in an indirect time domain dimension, t1(1Hα
/β
i−
1, 13Cα
/β
i−
1, 13Cα
i−
1, 13C′
i−
1).
- 1, and the chemical shift values for the following nuclei are measured;
-
40. The method according to claim 1, wherein N equals 5 and K equals 3 to conduct a (5,2)D [HBHACBCACA(CO)NHN] GFT NMR experiment, wherein (a) said sample is a protein molecule having two amino acid residues, i and i−
- 1, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
protons of amino acid residue i−
1, 1Hα
/β
i−
1;
(2) α
- and β
-carbons of amino acid residue i−
1, 13Cα
/β
i1;
(3) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(4) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 4 chemical shift evolution periods of the 5D FT NMR experiment, 1Hα
/β
i−
1, 13Cα
/β
i−
1, 13Cα
i−
1, and 15Ni, and (c) said jointly sampling comprises jointly sampling the 4 chemical shift evolution periods in an indirect time domain dimension, t1(1Hα
/β
i−
1, 13Cα
/β
i−
1, 13Cα
i−
1, 15Ni).
- 1, and the chemical shift values for the following nuclei are measured;
-
41. The method according to claim 1, wherein N equals 6 and K equals 4 to conduct a (6,2)D [HBHACBCACACONHN] GFT NMR experiment, wherein (a) said sample is a protein molecule having two amino acid residues, i and i−
- 1, and the chemical shift values for the following nuclei are measured;
(1) α
- and β
protons of amino acid residue i−
1, 1Hα
/β
i−
1;
(2) α
- and β
-carbons of amino acid residue i−
1, 13Cα
/β
i−
1;
(3) a polypeptide backbone carbonyl carbon of amino acid residue i−
1, 13C′
i−
1;
(4) a polypeptide backbone amide nitrogen of amino acid residue i, 15Ni; and
(5) a polypeptide backbone amide proton of amino acid residue i, 1HNi, (b) said selecting comprises selecting 5 chemical shift evolution periods of the 6D FT NMR experiment, 1Hα
/β
i−
1, 13Cα
/β
i−
1, 13Cα
i−
1, 13C′
i−
1, and 15Ni, and (c) said jointly sampling comprises jointly sampling the 5 chemical shift evolution periods in an indirect time domain dimension, t1(1Hα
/β
i−
1, 13Cα
/β
i−
1, 13Cα
i−
1, 13C′
i−
1, 15Ni).
- 1, and the chemical shift values for the following nuclei are measured;
-
42. The method according to claim 1, wherein N equals 5 and K equals 2 to conduct a (5,3)D [HCC,CH-COSY] GFT NMR experiment, wherein (a) the chemical shift values for the following nuclei are measured:
- (1) a proton, 1H;
(2) a carbon coupled to 1H, 13C; and
(3) a carbon coupled to 13C, 13Ccoupled; and
(4) a proton coupled to 13Ccoupled, 1Hcoupled, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 5D FT NMR experiment, 1H, 13C, and 13Ccoupled, and (c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(1H, 13C, 13Ccoupled).
- (1) a proton, 1H;
-
43. The method according to claim 42, wherein said chemical shift evolution periods for 13C and 13Ccoupled are correlated using total correlation spectroscopy (TOCSY).
-
44. The method according to claim 42, wherein (a) said sample is a protein molecule having an amino acid residue, i, and the chemical shift values for the following nuclei are measured:
- (1) a proton of amino acid residue i, 1Hi;
(2) a carbon of amino acid residue i coupled to 1Hi, 13Ci; and
(3) a carbon coupled to 13Ci, 13Cicoupled; and
(4) a proton coupled with 13Cicoupled, 1Hicoupled, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 5D FT NMR experiment, 1Hi, 13Ci, and 13Cicoupled, and (c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(1Hi, 13Ci, 13Cicoupled).
- (1) a proton of amino acid residue i, 1Hi;
-
45. The method according to claim 44, wherein said applying radiofrequency pulses comprises applying radiofrequency pulses for a 5D FT NMR experiment according to the scheme shown in FIG. 13.
-
46. The method according to claim 1, wherein N equals 5 and K equals 2 to conduct a (5,3)D [HBCBCGCDHD] GFT NMR experiment, wherein (a) said sample is a protein molecule having an amino acid residue, i, with an aromatic side chain, and the chemical shift values for the following nuclei are measured:
- (1) a β
-proton of amino acid residue i, 1Hβ
i;
(2) a β
-carbon of amino acid residue i, 13Cβ
i;
(3) a γ
-carbon of amino acid residue i, 13Cγ
i;
(4) a δ
-carbon of amino acid residue i, 13Cδ
i; and
(5) a δ
-proton of amino acid residue i, 1Hδ
i, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 5D FT NMR experiment, 1Hβ
i, 13Cβ
i, and 13Cδ
i, and (c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(1Hβ
i, 13Cβ
i, 13Cδ
i).
- (1) a β
-
47. The method according to claim 46, wherein said applying radiofrequency pulses comprises applying radiofrequency pulses for a 5D FT NMR experiment according to the scheme shown in FIG. 14.
-
48. The method according to claim 1, wherein N equals 5 and K equals 3 to conduct a (5,2)D [HBCBCGCDHD] GFT NMR experiment, wherein (a) said sample is a protein molecule having an amino acid residue, i, with an aromatic side chain, and the chemical shift values for the following nuclei are measured:
- (1) a β
-proton of amino acid residue i, 1Hβ
i;
(2) a β
-carbon of amino acid residue i, 13Cβ
i;
(3) a γ
-carbon of amino acid residue i;
(4) a δ
-carbon of amino acid residue i, 13Cδ
i; and
(5) a δ
-proton of amino acid residue i, 1Hδ
i, (b) said selecting comprises selecting 4 chemical shift evolution periods of the 5D FT NMR experiment, 1Hβ
i, 13Cβ
i, 13Cγ
i, and 13Cδ
i, and (c) said jointly sampling comprises jointly sampling the 4 chemical shift evolution periods in an indirect time domain dimension, t1(1Hβ
i, 13Cβ
i, 13Cγ
i, 13Cδ
i).
- (1) a β
-
49. The method according to claim 1, wherein N equals 4 and K equals 2 to conduct a (4,2)D [HCCH-COSY] GFT NMR experiment, wherein (a) the chemical shift values for the following nuclei are measured:
- (1) a proton, 1H;
(2) a carbon coupled to 1H, 13C;
(3) a carbon coupled to 13C, 13Ccoupled; and
(4) a proton coupled to 13Ccoupled, 1Hcoupled, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 4D FT NMR experiment, 1H, 13C, and 13Ccoupled, and (c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(1H, 13C, 13Ccoupled).
- (1) a proton, 1H;
-
50. The method according to claim 49, wherein said chemical shift evolution periods for 13C and 13Ccoupled are correlated using total correlation spectroscopy (TOCSY).
-
51. The method according to claim 49, wherein (a) said sample is a protein molecule having an amino acid residue, i, and the chemical shift values for the following nuclei are measured:
- (1) a proton of amino acid residue i, 1Hi;
(2) a carbon of amino acid residue i coupled to 1Hi, 13Ci;
(3) a carbon coupled to 13Ci, 13Cicoupled; and
(4) a proton coupled to 13Cicoupled, 1Hicoupled, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 4D FT NMR experiment, 1Hi, 13Ci, and 13Cicoupled, and (c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(1Hi, 13Ci, 13Cicoupled).
- (1) a proton of amino acid residue i, 1Hi;
-
52. The method according to claim 51, wherein said applying radiofrequency pulses comprises applying radiofrequency pulses for a 4D FT NMR experiment according to the scheme shown in FIG. 15.
-
53. The method according to claim 1, wherein N equals 5 and K equals 3 to conduct a (5,2)D [HCCCH-COSY] GFT NMR experiment, wherein (a) the chemical shift values for the following nuclei are measured:
- (1) a proton 1H;
(2) a carbon coupled to 1H, 13C;
(3) a carbon coupled to 13C, 13Ccoupled;
(4) a carbon coupled to 13Ccoupled, 13Ccoupled-2; and
(5) a proton coupled with 13Ccoupled-2, 1Hcoupled-2, (b) said selecting comprises selecting 4 chemical shift evolution periods of the 5D FT NMR experiment, 1H, 13C, 13Ccoupled, and 13Ccoupled-2, and (c) said jointly sampling comprises jointly sampling the 4 chemical shift evolution periods in an indirect time domain dimension, t1(1H, 13C, 13Ccoupled, 13Ccoupled-2).
- (1) a proton 1H;
-
54. The method according to claim 53, wherein (a) said sample is a protein molecule having an amino acid residue, i, and the chemical shift values for the following nuclei are measured:
- (1) a proton of amino acid residue i, 1Hi;
(2) a carbon of amino acid residue i coupled to 1Hi, 13Ci;
(3) a carbon coupled to 13Ci, 13Cicoupled;
(4) a carbon coupled to 13Cicoupled, 13Cicoupled-2; and
(5) a proton coupled with 13Cicoupled-2, 1Hicoupled-2, (b) said selecting comprises selecting 4 chemical shift evolution periods of the 5D FT NMR experiment, 1Hi, 13Ci, 13Cicoupled, and 13Cicoupled-2, and (c) said jointly sampling comprises jointly sampling the 4 chemical shift evolution periods in an indirect time domain dimension, t1(1Hi, 13Ci, 13Cicoupled, 13Cicoupled-2).
- (1) a proton of amino acid residue i, 1Hi;
-
55. The method according to claim 1, wherein N equals 5 and K equals 3 to conduct a (5,3)D [HCCCH-COSY] GFT NMR experiment, wherein (a) the chemical shift values for the following nuclei are measured:
- (1) a proton, 1H;
(2) a carbon coupled to 1H, 13C;
(3) a carbon coupled to 13C, 13Ccoupled;
(4) a carbon coupled to 13Ccoupled, 13Ccoupled-2; and
(5) a proton coupled with 13Ccoupled-2, 1Hcoupled-2, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 5D FT NMR experiment, 1H, 13C, and 13Ccoupled, and (c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(1H, 13C, 13Ccoupled).
- (1) a proton, 1H;
-
56. The method according to claim 55, wherein (a) said sample is a protein molecule having an amino acid residue, i, and the chemical shift values for the following nuclei are measured:
- (1) a proton of amino acid residue i, 1Hi;
(2) a carbon of amino acid residue i coupled to 1Hi, 13Ci;
(3) a carbon coupled to 13Ci, 13Cicoupled;
(4) a carbon coupled to 13Cicoupled, 13Cicoupled-2; and
(5) a proton coupled with 13Cicoupled-2, 1Hicoupled-2, (b) said selecting comprises selecting 3 chemical shift evolution periods of the 5D FT NMR experiment, 1Hi, 13Ci, and 13Cicoupled, (c) said jointly sampling comprises jointly sampling the 3 chemical shift evolution periods in an indirect time domain dimension, t1(1Hi, 13Ci, 13Cicoupled).
- (1) a proton of amino acid residue i, 1Hi;
Specification