1. Protein NMR Spectroscopy Institute of Biomedical Sciences
Yuan-Chao Lou
羅元超
Institute of Biomedical Sciences
Academia Sinica

2. Outline Classical Protein NMR Spectroscopy
(1). 2D HSQC spectra
(2). 3D Triple-Resonance Spectra
(3). Resonance Assignment
(4). Nuclear Overhauser Effect (NOE)
(5). Dihedral Angle and J Coupling Constant
(6). H-bond and Amide Proton Exchange Rate
B. Software for Structure Determination
(1). X-PLOR, CNS
(2). CYANA, Aria

3. 1D Protein NMR spectrum H2O Ha Ha side-chain protons Methyl protons
Aromatic protons Ha
Backbone NH Ha
Indo NH of Trp

4. 2D NOESY spectrum

5. Isotope Labeling
2D homonuclear NMR spectra are too crowded to make assignment when your protein size is more than 100 amino acids.
Stable isotope labeling (normally 15N and 13C) can provide additional dimensions to separate the signals.
Besides, the large coupling constants between these labeled nuclei facilitate the acquisition of triple resonance NMR spectra.

8. Why Bother with 15N Incorporation ?
15N-Heteronuclear Single Quantum Coherence (HSQC)
15N-1H

9. Why Bother with 13C Incorporation ?
13C-HSQC

10. 3D Triple-Resonance Spectroscopy

11. Coupling Constants between Nuclei
95 Hz O Cα Cβ Cγ N C’
140 Hz
11 Hz
55 Hz
30~40 Hz
15 Hz
~10 Hz H
13C
15N O
Magnetization transfer is via J-coupling interaction, so the larger the J-coupling, the more efficient the transfer. The sensitivity also depends on the T2 relaxation time of the nuclei.
140 Hz

12. Triple (1H, 13C, and 15N) Resonance Experiments
Through-bond experiments:
HNCA and HN(CO)CA
(b) HNCACB and CBCA(CO)NH
(c) HNCO and HN(CA)CO
(d) C(CO)NH and HCC(CO)NH
(e) HCCH-TOCSY

13. HNCA

14. HN(CO)CA

15. CBCA(CO)NH

16. HBHA(CO)NH

17. H(CC)(CO)NH

18. Sensitivity of Triple Resonance Experiments

19. 二項常用的三核共振實驗之脈衝圖譜

20. NMR Resonance Assignments Using Triple Resonance Experiments
Carry out HNCACB, CBCA(CO)NH, HNCO, and HNCACO.
HNCA and HN(CO)CA may also be needed.
(2) Sequential backbone assignments:
a. Identify those residues that have unique chemical shifts.
For example, Ca of Thr, Ile, Val, and Pro (> 60 ppm); Cb of
Thr, and Ser (> 60 ppm)
b. Ca of Ala (~ 50 ppm), Ca of Gly (~ 45ppm), and Cd of Pro (~ 50 ppm)
c. Cb of Leu, Asp, Asn, Ile, Phe, and Tyr (36~ 43ppm); Cb of Pro and
Val (30 ~ 35 ppm)
d. Others
(3) Using C(CO)NH or HCC(CO)NH etc to distinguish AMX residues from
the long-chain residues.

21. Sequential Assignment : From Ca and Cb resonances

22. Sequential Assignment :
From C’ resonances

23. Side-Chain
Resonance Assignment

24. Protein NMR Spectra

25. Chemical Shift Table of 20 Amino Acids

27. The Completeness of Assignment is an Determinant for NOESY Assignment
residue N C C C
other Q1
(8.379)
(4.111)
(2.834, 2.302)
C, (2.644, 2.644) D2
(7.959)
(4.746)
(3.154, 3.154) W3
(9.602)
(5.635)
(3.526, 3.317)
C1, (7.384); C3, (8.290); C2, (7.286); C2, (7.308); C3, (6.811); N1, (10.193) E4
(8.707)
(3.782)
(2.021, 2.021)
C, (2.422, 2.200) T5
(8.910)
(3.942)
(3.739)
C2, (1.248) F6
(8.796)
(4.228)
(3.615, 3.171)
C1, (7.165); C2, (7.165); C1, (7.024); C2, (7.024); C, (6.834) Q7
(8.118)
(3.647)
(1.193, 1.193)
C, (1.851, 1.851); N2, (6.195, 4.472) K8
(7.449)
(4.036)
(1.807, 1.770)
C, (1.487, 1.487); C, (1.710, 1.710); C, (2.944, 2.944) K9
(8.261)
(4.167)
(1.626, 1.626)
C, (1.090, 1.090); C, (1.398, 1.398); C, (2.882, 2.882)
H10
(7.803)
(4.846)
(2.767, 2.000)
C2, (6.786); C1, (8.755)

28. NOESY spectra

29. Nuclear Overhauser Effect
Nuclear Overhauser effect (NOE) was discovered by Albert Overhauser in He found that saturation of the electron magnetic resonance in a paramagnetic system would cause the nuclear resonance intensity to be enhanced. A similar effect occurs between nuclei. It is much smaller, but still observable.
NOE enhancements between nuclei are due to nuclei’s dipole-dipole interactions (through-space) and are correlated with the inverse sixth power of the internuclear distance.
 : permeability constant
h : Planck’s constant
 : magnetogyric ratio
c : rotational correlation time
 : larmor frequency
J() = 2c / (1 + 2c2)

30. Dependence of NOE on Molecular Motion
If I = S , NOE = 0 when c = 1.12
A peptide which contains around 10 residues can only get very weak NOE signals.

31. Dependence of NOE on Distance
NOE / NOEstd
= rstd6 / r 6
The shorter the distance, the stronger the NOE effect. Basically, we can observe the NOE cross peak between two protons if their distance is smaller than 5 Å. And we can get the distance information from the intensity of the NOE cross-peaks.

32. Dihedral Angles Cγ χ2 Cβ χ1 Cα N ψ Ψ ω N C’ O
Karplus Equations:

33. Dihedral Angles and J Coupling Constants of Different Secondary Structures
Dihedral Angles (deg)
3JHNα(HZ)
≧ 9
≦ 4 β βp
α-helix
310-helix
π-helix
Polyproline I
Polyproline II
Polyglycine II ψ
-139
-119
-57
-49
-83
-78
-80 Ψ
+135
+113
-47
-26
-70
+158
+149
+150 ω
-178
180
Adapted from G. N. Ramachandran and V. Sasisekharan, Adv. Protein Chem. 23, (1968); IUPAC-IUB Commission on biochemical Nomemclature, Biochemistry 9, (1970).

34. Extracting J Coupling Constants from 1D spectra
3JHNα

35. Extracting J Coupling Constants from 2D DQF-COSY
3JHNα

36. Extracting J Coupling Constants from 3D HNHA Spectra
Scross/Sdiag = -tan2(2JHNa )

37. Getting Dihedral Angle Restraints from Searching a Database by TALOS
TALOS is a database system for empirical prediction of phi (f) and psi (y) backbone dihedral angles using a combination of five kinds (Ha, Ca, Cb, C’, N) of chemical shift assignments for a given protein sequence.

38. Getting H-bond Restraints from Amide Proton Exchange Rate
Exchangeable Protons:
－NH ; －OH ; －SH
Amide protons that are protected by H-bonds or hydrophobic residues exhibit lower exchange rate

39. Proton Distances, Coupling Constants, and Amide Proton Exchange rate in a-helix
Parameter
dαN(i,i+1)
dαN(i,i+2)
dαN(i,i+3)
dαN(i,i+4)
dNN(i,i+1)
dNN(i,i+2)
dβN(i,i+1)
dαβ (i,i+3)
3JHNα(HZ)
HN exchange rate
α-helix
3.5
4.4
3.4
4.2
2.8
(≦4)
slow
310-helix
3.4
3.8
3.3
(>4.5)
2.6
4.1
(≦4)
slow C’ N’
The first four residues in the α-helix and the first three residues in the 310-helix will have fast amide proton exchange rates.

40. Proton Distances, Coupling Constants, and Amide Proton Exchange rate in b-strand
Parameter
dαN(i,i)
dαN(i,i+1)
dNN(i,i+1)
dβN(i,i+1)
dαα (i,j)
dαN (i,j)
dNN (i,j)
3JHNα(HZ)
NH exchange rate β
2.8
2.2
4.3
2.3
3.2
3.3
(≧9)
slow βP
2.8
2.2
4.2
4.8
3.0
4.0
(≧9)
slow N’ C’ N’ C’
dαα(i,j), dαN (i,j) and dNN (i,j) refer to interstrand distances.
Every second residue in the flanking strand will have slow amide proton exchange rates

41. Observed NOEs in Secondary Structures
dNN(i,i+1)
dαN(i,i+1)
dαN(i,i+3)
dαβ (i,i+3)
dαN(i,i+2)
dNN(i,i+2)
dαN(i,i+4)
3JHNα(HZ)
α-helix
310-helix
β-strand
The thickness of the lines is an indication of the intensity of the NOEs
The values of J coupling are approximate.

42. TOCSY and NOESY Spectra of Tc1

43. TOCSY : Amide to Aliphatic Region
N’-ACGSC RKKCK GSGKC INGRC KCY-C’

44. NOESY and TOCSY : Amide to a RegionH O

45. NOESY Spectra of TC1
Fingerprint and NH/NH regions of 400ms NOESY spectra of 1mM Tc1 in 90% H2O /10% D2O at 275K, pH 3.0

46. The Definition of b -sheet of TC1
The b-sheet structure of Tc1 can be defined based on NOEs and amide proton exchange rate

47. Summary of the amide proton exchange rates, 3JNHα coupling constants, NOE connectivities, chemical shift index, and the derived secondary structures

48. Protein NMR Structure Determination
Protein in solution
~0.3 ml, 0.5 mM concentration
Sample preparation:
cloning,
protein expression
purification,
characterization,
isotopic labeling.
Distances between
protons (NOE),
Dihedral angles(J
coupling), H-bond
(Amide-proton
exchange rate ),
RDC restraints
NMR spectroscopy
1D, 2D, 3D, …
Secondary
structure of
protein
Sequence-specific
Resonance assignment
Extraction of Structural
information
Structure calculation
Final 3D
structures
Structure refinement

49. The Completeness of Assignment is an Determinant for NOESY Assignment
residue N C C C
other Q1
(8.379)
(4.111)
(2.834, 2.302)
C, (2.644, 2.644) D2
(7.959)
(4.746)
(3.154, 3.154) W3
(9.602)
(5.635)
(3.526, 3.317)
C1, (7.384); C3, (8.290); C2, (7.286); C2, (7.308); C3, (6.811); N1, (10.193) E4
(8.707)
(3.782)
(2.021, 2.021)
C, (2.422, 2.200) T5
(8.910)
(3.942)
(3.739)
C2, (1.248) F6
(8.796)
(4.228)
(3.615, 3.171)
C1, (7.165); C2, (7.165); C1, (7.024); C2, (7.024); C, (6.834) Q7
(8.118)
(3.647)
(1.193, 1.193)
C, (1.851, 1.851); N2, (6.195, 4.472) K8
(7.449)
(4.036)
(1.807, 1.770)
C, (1.487, 1.487); C, (1.710, 1.710); C, (2.944, 2.944) K9
(8.261)
(4.167)
(1.626, 1.626)
C, (1.090, 1.090); C, (1.398, 1.398); C, (2.882, 2.882)
H10
(7.803)
(4.846)
(2.767, 2.000)
C2, (6.786); C1, (8.755)
L11
(8.311)
(5.406)
(2.156, 2.156)
C, (1.788); C1, (1.103); C2, (1.103)
T12
(8.237)
(5.003)
(3.775)
C2, (1.220)
D13
(8.271)
(4.874)
(3.060, 2.766)
T14
(8.106)
(4.802)
(4.067)
C2, (0.947)
K15
(8.239)
(3.623)
(1.440, 1.440)
C, (0.736, 0.405); C, (1.423, 1.423); C, (2.741, 2.741)
K16
(7.904)
(4.277)
(1.680, 1.680)
C, (1.234, 1.234); C, (1.545, 1.545); C, (2.953, 2.953)
V17
(6.052)
(3.325)
(1.441)
C1, (0.405); C2, (0.105)
K18
(8.665)
(4.488)
(1.886, 1.886)
C, (1.548, 1.548); C, (1.748, 1.748); C, (3.062, 3.062)
C19
(8.066)
(3.692)
(3.027, 2.339)
D20
(8.880)
(4.453)
(3.056, 2.905)

50. NOESY Assignment
10A
10B
10C
Intra-residual NOE ( |i-j| = 0)
Sequential NOE ( |i-j| = 1)
Medium-range NOE ( |i-j| < 5)
Long-Range NOE ( |i-j| ≥ 5) C 3B 1 D A B 1B 1C 1D 2 B 3 B 4 2B 5 D C 6 C 11 B B 1 A 10 7 A 2 9 8 B 8 3 B 9 4 7 B C 10 A 5 6 11 1A

51. Restraints for Structure Calculation
NOE restraints :
assign (resid 9 and name HN ) (resid 6 and name HA )
assign (resid 9 and name HN ) (resid 8 and name HA )
assign (resid 9 and name HN ) (resid 9 and name HA )
Dihedral Angle Restraints:
! phi residue 9
assign (resid 8 and name C ) (resid 9 and name N )
(resid 9 and name CA) (resid 9 and name C )
! psi residue 9
assign (resid 9 and name N) (resid 9 and name CA)
(resid 9 and name C) (resid and name N )
H-bond Restraints:
assign (resid 9 and name HN) (resid 5 and name O )
assign (resid 9 and name N ) (resid 5 and name O )

52. Three-Dimensional Structure Determination by Simulated Annealing using X-PLOR or CNS Program
dih
noe
vdw
improper
angle
bond
total E + =
Keep the correctness of protein geometry
The energy terms of experimental data

53. Automated NOESY Assignment and Structure Calculation
Protein Sequence
Chemical shift list
Positions and volumes of NOESY cross peaks
Automated methods are
- much faster
- more objective
Find new NOE assignments
Problems may arise because of
- imperfect input data
- limitation of the algorithms used
Structure Calculation
Iterative process : All but the first cycle use the structure from the preceding cycle.
Evaluate Assignments
Finish NOESY Assignment
3D NMR Structure
The first cycle is important for the reliability of the method.

54. Algorithms Used by CYANA
Network-Anchoring can find the new NOE assignment correctly.

55. Structural Statistics of the Best 20 Structures

56. Ramachandran Plot