Phase sensitively-detected reduced dimensionality nuclear magnetic resonance spectroscopy for rapid chemical shift assignment and secondary structure determination of proteins
View Patent ↗The present invention discloses eleven reduced dimensionality (RD) triple resonance nuclear magnetic resonance (NMR) experiments for measuring chemical shift values of certain nuclei in a protein molecule, where the chemical shift values encoded in a peak pair of an NMR spectrum are detected in a phase sensitive manner. The RD 3D HA , CA ,(CO),N,HN NMR and RD 3D H , C ,(C-TOCSY-CO),N,HN NMR experiments are designed to yield “sequential” connectivities, while the RD 3D H α/β , C α/β ,CO,HA NMR and RD 3D H α/β , C α/β ,N,HN NMR experiments provide “intraresidue” connectivities. The RD 3D H , C ,C,H-COSY NMR, RD 3D H , C ,C,H-TOCSY NMR, and RD 2D H , C ,H-COSY NMR experiments allow one to obtain assignments for aliphatic and aromatic side chain chemical shifts, while the RD 2D HB , CB ,(CG,CD),HD NMR experiment provide information for the aromatic side chain chemical shifts. In addition, methods of conducting suites of RD triple resonance NMR experiments for high-throughput resonance assignment of proteins and determination of secondary structure elements are disclosed.
1. A method for obtaining assignments of chemical shift values of 1 H, 13 C and 15 N of a protein molecule comprising:
providing a 15 N/ 13 C-labeled protein sample; and
conducting four reduced dimensionality (RD) nuclear magnetic resonance (NMR) experiments on the protein sample, wherein the chemical shift values of 1 H and 13 C which are encoded in peak pairs of an NMR spectrum are detected in a phase sensitive manner and (1) a first experiment is selected from the group consisting of a RD three-dimensional (3D) H α/β C α/β (CO)NHN NMR experiment, a RD 3D HA , CA ,(CO),N,HN NMR experiment, and a RD 3D H , C ,(C-TOCSY-CO),N,HN NMR experiment for obtaining sequential correlations of chemical shift values; (2) a second experiment is selected from the group consisting of a RD 3D HNN CAHA NMR experiment, a RD 3D H α/β , C α/β ,N,HN NMR experiment, and a RD 3D HNN< CO , CA > NMR experiment for obtaining intraresidue correlations of chemical shift values; (3) a third experiment is a RD 3D H , C ,C,H-COSY NMR experiment for obtaining assignments of sidechain chemical shift values; and (4) a fourth experiment is a RD two-dimensional (2D) HB , CB ,(CG,CD),HD NMR experiment for obtaining assignments of aromatic sidechain chemical shift values.
2. The method according to claim 1 further comprising:
subjecting the protein sample to a RD 2D H , C ,H-COSY NMR experiment for obtaining assignments of sidechain chemical shift values.
3. The method according to claim 1 , wherein the first experiment is the RD 3D H α/β C α/β (CO)NHN NMR experiment and the second experiment is the RD 3D HNN CAHA NMR experiment.
4. The method according to claim 3 further comprising:
subjecting the protein sample to a RD 3D HA , CA ,(CO),N,HN NMR experiment to distinguish between NMR signals for 1 H α / 13 C α , and 1 H β / 13 C β from said RD 3D H α/β C α/β (CO)NHN NMR experiment.
5. The method according to claim 3 further comprising:
subjecting the protein sample to a RD 3D H , C ,(C-TOCSY-CO),N,HN NMR experiment to obtain assignments of chemical shift values of 1 H ali and 13 C ali .
6. The method according to claim 3 further comprising:
subjecting the protein sample to a RD 3D H α/β , C α/β ,N,HN NMR experiment to obtain assignments of chemical shift values of 1 H β and 13 C β .
7. The method according to claim 3 further comprising:
subjecting the protein sample to a RD 3D HNN< CO , CA > NMR experiment to obtain assignments of chemical shift values of polypeptide backbone carbonyl carbons, 13 C′.
8. The method according to claim 3 further comprising:
subjecting the protein sample to a RD 3D H α/β , C α/β ,CO,HA NMR experiment to obtain assignments of chemical shift values of polypeptide backbone carbonyl carbons, 13 C′.
9. The method according to claim 3 further comprising:
subjecting the protein sample to a RD 3D HNN< CO , CA > NMR experiment and a RD 3D H α/β , C α/β ,CO,HA NMR experiment to obtain assignments of chemical shift values of 13 C′.
10. The method according to claim 3 further comprising:
subjecting the protein sample to a RD 3D H , C ,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of 1 H and 13 C of aliphatic sidechains.
11. The method according to claim 3 further comprising:
subjecting the protein sample to a RD 3D H , C ,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of 1 H and 13 C of aromatic sidechains.
12. The method according to claim 3 further comprising:
subjecting the protein sample to a 3D HNNCACB NMR experiment to obtain assignments of chemical shift values of 13 C β .
13. The method according to claim 3 , wherein the first experiment is the RD 3D H , C ,(C-TOCSY-CO),N,HN NMR experiment and the second experiment is the RD 3D HNN CAHA NMR experiment.
14. The method according to claim 13 further comprising:
subjecting the protein sample to a RD 3D HA , CA ,(CO),N,HN NMR experiment to identify NMR signals for 1 H α / 13 C α in said RD 3D H , C ,(C-TOCSY-CO),N,HN NMR experiment.
15. The method according to claim 13 further comprising:
subjecting the protein sample to a RD 3D H α/β , C α/β ,N,HN NMR experiment to obtain assignments of chemical shift values of 1 H β and 13 C β .
16. The method according to claim 13 further comprising:
subjecting the protein sample to a RD 3D HNN< CO , CA >NMR experiment to obtain assignments of chemical shift values of polypeptide backbone carbonyl carbons, 13 C′.
17. The method according to claim 13 further comprising:
subjecting the protein sample to a RD 3D H α/β , C α/β ,CO,HA NMR experiment to obtain assignments of chemical shift values of polypeptide backbone carbonyl carbons, 13 C′.
18. The method according to claim 13 further comprising:
subjecting the protein sample to a RD 3D HNN< CO , CA >NMR experiment and a RD 3D H α/β , C α/β ,CO,HA NMR experiment to obtain assignments of chemical shift values of 13 C′.
19. The method according to claim 13 further comprising:
subjecting the protein sample to a RD 3D H , C ,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of 1 H and 13 C of aliphatic sidechains.
20. The method according to claim 13 further comprising:
subjecting the protein sample to a RD 3D H , C ,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of 1 H and 13 C of aromatic sidechains.
21. The method according to claim 13 further comprising:
subjecting the protein sample to a 3D HNNCACB NMR experiment to obtain assignments of chemical shift values of 13 C β .
22. The method according to claim 1 , wherein the first experiment is the RD 3D H , C ,(C-TOCSY-CO),N,HN NMR experiment and the second experiment is the RD 3D H α/β , C α/β ,N,HN NMR experiment.
23. The method according to claim 22 further comprising:
subjecting the protein sample to a RD 3D HA , CA ,(CO),N,HN NMR experiment to identify NMR signals for 1 H α and 13 C α in said RD 3D H , C ,(C-TOCSY-CO),N,HN NMR experiment.
24. The method according to claim 22 further comprising:
subjecting the protein sample to a RD 3D H α/β C α/β (CO)NHN NMR experiment to identify NMR signals for 1 H α/β and 13 C α/β in said RD 3D H , C ,(C-TOCSY-CO),N,HN NMR experiment.
25. The method according to claim 22 further comprising:
subjecting the protein sample to a RD 3D HNN< CO , CA >NMR experiment to obtain assignments of chemical shift values of polypeptide backbone carbonyl carbons, 13 C′.
26. The method according to claim 22 further comprising:
subjecting the protein sample to a RD 3D H α/β , C α/β ,CO,HA NMR experiment to obtain assignments of chemical shift values of polypeptide backbone carbonyl carbons, 13 C′.
27. The method according to claim 22 further comprising:
subjecting the protein sample to a RD 3D HNN< CO , CA >NMR experiment and a RD 3D H α/β , C α/β ,CO,HA NMR experiment to obtain assignments of chemical shift values of 13 C′.
28. The method according to claim 22 further comprising:
subjecting the protein sample to a RD 3D H , C ,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of 1 H and 13 C of aliphatic sidechains.
29. The method according to claim 22 further comprising:
subjecting the protein sample to a RD 3D H , C ,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of 1 H and 13 C of aromatic sidechains.
30. The method according to claim 22 further comprising:
subjecting the protein sample to a 3D HNNCACB NMR experiment to obtain assignments of chemical shift values of 13 C β .
31. The method according to claim 1 , wherein the first experiment is the RD 3 H , C ,(C-TOSY-CO),N,HN NMR experiment and the second experiment is the RD 3D HNN< CO , CA ,>NMR experiment.
32. The method according to claim 31 further comprising:
subjecting the protein sample to a RD 3D HA , CA ,(CO),N,HN NMR experiment to identify NMR signals for 1 H α in said RD 3D H , C ,(C-TOCSY-CO),N,HN NMR experiment.
33. The method according to claim 31 further comprising:
subjecting the protein sample to a RD 3D H α/β C α/β (CO)NHN NMR experiment to identify NMR signals for 1 H α/β and 13 C α/β in said RD 3D H , H (C-TOCSY-CO),N,HN NMR experiment.
34. The method according to claim 31 further comprising:
subjecting the protein sample to a RD 3D H α/β , C α/β ,CO,HA NMR experiment to obtain assignments of chemical shift values of polypeptide backbone carbonyl carbons, 13 C′.
35. The method according to claim 31 , further comprising:
subjecting the protein sample to a RD 3D H , C ,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of 1 H and 13 C of aliphatic sidechains.
36. The method according to claim 31 further comprising:
subjecting the protein sample to a RD 3D H , C ,C,H-TOCSY NMR experiment to obtain assignments of chemical shift values of 1 H and 13 C of aliphatic sidechains.
37. The method according to claim 31 further comprising:
subjecting the protein sample to a RD 3D HNNCACB NMR experiment to obtain assignments of chemical shift values of 13 C β .
38. The method according to claim 31 further comprising:
subjecting the protein sample to a nuclear Overhauser effect spectroscopy (NOESY) to deduce the tertiary structure of the protein molecule.
39. The method according to claim 31 further comprising:
subjecting the protein sample to NMR experiments that measure scalar coupling constants to deduce the tertiary structure of the protein molecule.
40. The method according to claim 22 further comprising:
subjecting the protein sample to a 3D HNNCACB NMR experiment to obtain assignments of chemical shift values of 13 C β .