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Resonance assignment strategies

Resonance assignment strategies. The assignment problem. Amino acid sequence. +. A ssignment via 1 H NMR. Proton frequencies. Spin Systems. - Assignment based on backbone H N ➡ present in all residues (except Proline) ➡ unique region of spectrum ➡ well-dispersed resonances

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Resonance assignment strategies

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  1. Resonance assignment strategies

  2. The assignment problem Amino acid sequence +

  3. Assignment via 1H NMR

  4. Proton frequencies

  5. Spin Systems • - Assignment based on backbone HN • ➡ present in all residues (except Proline) • ➡ unique region of spectrum • ➡ well-dispersed resonances • -Scalar couplings (COSY / TOCSY) • ➡ identify spin systems (i.e. amino acid type) • - number of resonances (i.e. protons) • frequency of resonances • -Connect with NOESY

  6. intra-residual NOEs sequential NOEs 1H - 1H NOE • Dipolar interaction (NOEs) • through-space contacts • intra-residual, sequential (& long-range) contacts • link spin-systems: identify i & i-1 residue "i -1" residue "i" residue "i+1"

  7. 1 2 3 4 5 6 7 8 9 Intra residue Inter residue Example Peptide sequence R1-A2-Q3-L4-A5-M6-S7

  8. 1 2 3 4 5 6 7 8 9 Intra residue Inter residue ??? Example: who is who? Peptide sequence R1-A2-Q3-L4-A5-M6-S7

  9. 1 2 3 4 5 6 7 8 9 Intra residue Inter residue Example: identify protons & frequencies Peptide sequence R1-A2-Q3-L4-A5-M6-S7

  10. 1 2 3 4 5 6 7 8 9 Intra residue Inter residue Example: assign strips to residues Peptide sequence R1-A2-Q3-L4-A5-M6-S7 C = Ala

  11. 1 2 3 4 5 6 7 8 9 Intra residue Inter residue Example: assign strips to residues Peptide sequence R1-A2-Q3-L4-A5-M6-S7 B = Leu C = Ala

  12. 1 8 2 3 9 5 6 7 4 Intra residue Inter residue Example: assign strips to sequence Peptide sequence R1-A2-Q3-L4-A5-M6-S7 Possibilities I: A2 - Q3 - L4 (CAB) II: Q3 - L4 - A5 (ABC) III: L4 - A5 - M6 (BCA) A = Q3 / M6 B = L4 C = A2 / A5

  13. 1 2 3 4 5 6 7 8 9 Example: connect residues • Use HN-HN NOEs • B has cross-peaks to both A & C • ABC • Q3 - L4 - A5 Possibilities I: A2 - Q3 - L4 (CAB) II: Q3 - L4 - A5 (ABC) III: L4 - A5 - M6 (BCA) Peptide sequence R1-A2-Q3-L4-A5-M6-S7

  14. 1 2 3 4 5 6 7 8 9 Example: verify!!! • Use HN-Hα NOEs to verify • sequential HN(i) - Hα(i-1) • HN(C) - Hα(B) • HN(B) - Hα(A) • ABC • Q3 - L4 - A5 Possibilities I: A2 - Q3 - L4 (CAB) II: Q3 - L4 - A5 (ABC) III: L4 - A5 - M6 (BCA) Peptide sequence R1-A2-Q3-L4-A5-M6-S7

  15. Assignment via 1H, 15N, and 13C

  16. J coupling constants

  17. Triple resonance NMR • Heteronuclear experiments • more information • increase resolution: 2D → 3D → 4D ... • sequential assignment based on scalar coupling Protons Other nuclei 13C, 15N • Advantages • through-bond (J) magnetization transfer to neighboring residues (instead of NOE) • 1J scalar coupling much larger than 3JHH (<10 Hz) (efficient transfer of magnetization)

  18. Nomenclature i-1 & i i-1 Residue i-1 & i i-1 HN(CA)CO HNCO HNCA HN(CO)CA i-1 & i i-1 Names of scalar experiments based on atoms detected Pairs of experiments distinguish between intra-residual and sequential resonances HN(CA)CB HN(COCA)CB

  19. Example: 3D HNCA

  20. O O O O O O R R R R R R --15N–13Cα–13C–15N–13Cα–13C-- --15N–13Cα–13C–15N–13Cα–13C-- --15N–13Cα–13C–15N–13Cα–13C-- H H H H H H H H H H H H Example: analyze frequencies a b 122.8 123.8 68.43 61.32 61.32 58.52 7.71 8.40 117.1 55.03 68.43 c 8.24

  21. Example: link the spin-systems • Numerically... • c: Cα (i) = a: Cα (i-1) • a: Cα (i) = b: Cα (i-1) Sequence: c – a – b

  22. Example: link the spin-systems

  23. HNCA versus HN(CO)CA i & i-1 15N 13Cα 1HN i-1

  24. Assigned [1H-15N]-HSQC 15N 1H

  25. If no label or only 15N: NOESY / TOCSY Identify spin-system in TOCSY Sequential NOEs to link spin-systems 13C & 15N: 3D triple resonance experiments Sequential information through bond (J coupling) HNCA / HN(CO)CA (and many more) Key concepts assignment

  26. NMR observables&structural restraints

  27. Protein structure • Secondary structure • alpha helix, beta-sheet, etc. • Tertiary structure • full 3D structure • Experimental data that give information about the protein structure • NMR observables • Translate the experimental data into parameters that can be used in a structure calculation • Structural restraints

  28. NMR observables vs. structural restraints -3J-coupling dihedral angle - Chemical shifts secondary structure - NOE’s H-H distances - Paramagnetic relaxation enhancement (PRE) distances - Residual dipolar coupling (RDC) orientation of vectors - H/D exchange hydrogen bonds

  29. measured 3J(HNHα) reports on φ OBSERVABLE: homonuclear J-couplings φ φ Karplus relation: J = A.cos2(φ) + B.cos (φ) + C

  30. RESTRAINTS: dihedral angles ω ~ 180º C C C C C N N C φ ψ ω O O

  31. OBSERVABLE: chemical shift • 13Cα and 13Cβ chemical shifts • sensitive to dihedral angles • report on secondary structure elements

  32. Chemical Shift Index (CSI)

  33. Predicting dihedral angels: TALOS

  34. ψ φ φ ψ RESTRAINTS: dihedral angles anti-parallel β-strand α-helix β-strand α-helix φ -130 -60 ψ 125 -45

  35. β-strand α-helix Ramachandran plot +180 ψ -180 -180 φ +180

  36. Longe range • • • • A B C D Z Medium range OBSERVABLE: NOE r = • 1H-1H NOEs • signal intensity proportional to 1/r6 • reports on distance between protons • distance restraints (up to 5-6 Å) r = Sequential Intra-residue (used for identifying spin-systems) Sequential & medium range NOEs - SECONDARY STRUCTURE

  37. RESTRAINT: distances

  38. NOEs in secondary structure elements

  39. Short distances in β-strands anti-parallel

  40. NOEs in secondary structure elements

  41. Short distances in α-helices

  42. Short distances in α-helices

  43. OBSERVABLE: PRE • paramagnetic relaxation enhancement (PRE) • paramagnetic center (unpaired electron) • radical (e.g.nitroxide) • certain metal ions (i.e. Mn2+, Gd3+) • nuclear spin relaxation is enhanced by the paramagnetic center • signals will broaden (or even disappear) • effect is dependent on the distance to the paramagnetic center • 1/r6 • because of the large magnetic moment of the unpaired electron the PRE provides long-range distance information (Mn2+ ~35 Å)

  44. OBSERVABLE: Residual dipolar couplings Dipolar coupling

  45. Residual dipolar coupling (RDC) Dipolar coupling

  46. Residual dipolar coupling (RDC)

  47. RDC: Orientational restraint RDC reports on orientation of bond-vector - orientation of bond-vector within a molecular alignment tensor (defined by Aa and Ar) with respect to the magnetic field Long range orientational restraint - TERTIARY STRUCTURE

  48. The more RDCs, the better... • RDCs commonly measured • 1D 1HN-15N • 1D 13C’-15N • 1D 13Cα-13C’ • 1D 1Hα-13Cα • In perdeuterated proteins • 2D 1HN-13C’ • 2D/3D 1HN-13Cα C C C C C N N C O O

  49. RESTRAINT: RDC Orientation

  50. OBSERVABLE: H/D exchange rates

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