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Chem-806 Identification of organic and inorganic compounds by advance NMR techniques

Chem-806 Identification of organic and inorganic compounds by advance NMR techniques. Tool box 2D-NMR: Homonuclear 2D-NMR: Heteronuclear 3D-NMR. NMR of protein. 1 H NMR spectra of small protein. Protein : folded and unfolded. Folded. Unfolded. 2D-COSY. 2D-COSY: NH/CH a expanded region.

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Chem-806 Identification of organic and inorganic compounds by advance NMR techniques

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  1. Chem-806Identification of organic and inorganic compounds by advance NMR techniques Tool box 2D-NMR: Homonuclear 2D-NMR: Heteronuclear 3D-NMR

  2. NMR of protein

  3. 1H NMR spectra of small protein

  4. Protein : folded and unfolded Folded Unfolded

  5. 2D-COSY

  6. 2D-COSY: NH/CHa expanded region

  7. 3JHH and dihedral angle H H H H Gauche and trans conformation can be distinguished by measuring coupling

  8. 2D-NOESY

  9. sequential assignment using NOESY • In the α-helix the neighbouring HN are 2.8 Ångström apart • In the β-strand the distance from Hα in residue (i) to HN in residue (i+1) is only 2.2 Ångström.

  10. Schematic presentation : NOESY spectrum in the NH region The diagonal cross peaks are marked as black signals labeled 1 to 9. The red cross peaks are sequential NOEs. If 2=Val (V), 5 from a tryptophan (W) and 9 from a glycine (G), we can write that sequence is XVXXXWXXG or GXXWXXXVX 983567421 sequence of signals GAKWSRYVP amino acid sequence 1  2  4  7  6  5  3  8  9 Or reverse

  11. Sequential Assignment of Hαin residue (i) to NHin residue (i+1) by NOE Superimposition of a COSY spectrum with blue annotated cross peaks(through bond) and NOESY spectrum with red cross peaks. Sequential NOE between Hα in residue (i) to HN in residue (i+1). CH1/NH5 6  4  1 5  7  3  2 CH4/NH1 CH6 NH4 NH6

  12. NOE-distance using distance constraints to calculate a structure. In the upper figure is shown a linear strand with beads. The green, red and blue pairs of beads, respectively have been shown to be close to each other. The structure below represents one solution to determining the structure based on the three pieces of distance information.

  13. Coupling values to setup experiments 140 Hz H 15 Hz 11 Hz 55 Hz 13C 13C 15N 15N 13C O O H H 13C 90-100 Hz 30-40 Hz

  14. Some 3D sequences

  15. 3D-HMQC-COSY

  16. t3 t t t2 mix 1H t1 13C t t1 = Ca t2 = Ha t3 = HNOE = 1 / 2JCH F1 d13Ca 1 2 3 F2 dHa dNH F3 HMQC-NOESY NH 1 2 Ha1 Ca1 3 2 Ha2 Ca2 3 4 Ca3

  17. HMQC-NOESY R. R. Ernst (nobel lecture ‘92)

  18. 3D-HMQC-NOESY

  19. 2D-NOESY vs 3D-HMQC-NOESY

  20. 90 180 90 45 45 t3 t1 = 1H t1 t mix 1H t2 = 15N D t2 D 15N t3 = NH D = 1 / 2JNH ~4.5 ms F2 t = 1 / 2dH2O water suppression d15H 1 2 3 F1 dHa dNH F3 NOESY-HMQC Ha-1 Ha1 NH1 Ha2 NH2 Ha1 Ha3 Ha2 NH3 L.E.Kay, D.Marion, A.Bax, J.Magn.Reson.,84, 72 (89)

  21. NOESY-HMQC and HMQC-NOESY

  22. GE-HMQC-TOCSY

  23. 2D and 3D HMQC-TOCSY

  24. 3D HCCH-TOCSY

  25. 3D-NMR: Puzzle approach HNCA HN(CO)CA HNCO

  26. 3D-NMR: Puzzle approach HA(CA)NH HACACO HCA(CO)N

  27. HNCACB CBCA(CO)NH

  28. Figure 19. Schematic presentation of the combined spectrum analysis of the three-dimensional HNCACB and a CBCA(CO)NH spectra. The 15N axis and the frames in the 15N dimension are coloured blue. The1H axis and the frames in the 1H dimension are coloured red. The 13C axis and the frames in the 13C dimension are green. Two planes δN(i), top-left, and δN(i+1), bottom-left, are highlighted. The cross peaks in HNCACB are • and in CBCA(CO)NH are •

  29. {H2O} t3 d d t t t t 1H T t1/2 t1/2 15N t2 13C=O t = 1 / 4JNH ~ 2.25 ms 13Ca HNCO : Sequential assignment in protein 1JNH ~ 90-100 Hz 1JN-13C=O ~ 15 Hz 1JN-13Ca ~ 11 Hz t1 = 15N t2 = C=O t3 = HN d = 18 ms Bax et all., J.Magn.Reson., 89, 486-514 (90)

  30. HNCO 2D and 3D

  31. t = 1 / 4JNH ~ 2.25 ms HNCA : Sequential assignment in protein 1JNH ~ 90-100 Hz 1JN-13C=O ~ 15 Hz 1JN-13Ca ~ 11 Hz 2JN-13Ca-1 ~ 7 Hz t3 t t t t 1H t1/2 t1/2 15N d d t2 13Ca 13C=O t2 = Ca t1 = 15N t3 = HN d= 1 / 2 JN-Ca ~ 33 ms

  32. t = 1 / 4JCH ~ 1.5 ms HCACO : Sequential assignment in protein 1JCH ~ 140 Hz 1J13Ca-13C=O ~ 55 Hz t3 t t t t 1H t1/2 13Ca t1/2 D D COSY t2 13C=O 15N t1 = Ca t3 = H Ca t2 = 13C=O D= 1 / 4 JCa-CO ~ 3 ms

  33. t = 1 / 4JCH ~ 1.5 ms HCA(CO)N : Sequential assignment in protein 1JCH ~ 140 Hz 1J13Ca-13C=O ~ 55 Hz 1J15N -13C=O ~ 15 Hz t3 t t t t 1H t1/2 13Ca t1/2 D D COSY 13C=O d d t2 15N t1 = Ca t3 = H Ca t2 = 15N D= 1 / 4 JCa-CO ~ 3 ms d= 1 / 3 JCO-N ~ 18-20 ms

  34. HN(CA)CO and HNCOcomplementary experiments

  35. Example of HNCA and HN(CO)CA

  36. Example of sequential assignment

  37. HN(CO)(CA)

  38. 2D HN(CO)(CA) NH Leu-6 NH Phe-4 NH Leu-3 NH Ala-5 NH ala-2 NH

  39. 4D-NMR

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