1. Protein Structure Determination by NMR Spectroscopy

2. 1D Spectrum
amide/
aromatic/
region
alpha
region
methyl
region

3. 0.015 M glucagon 360 MHz

4. 0.01 M Inhibitor K , 360 MHz

6. Relayed COSY. (with one homonuclear relayed).
2D J-resolved
COSY 45. COSY 90. COSY DQF. TOCSY.
COSY L.R. TOCSY
Relayed COSY. (with one homonuclear relayed).
NOESY. ROESY.

7. Protein Structure Determined by NMR
1D NMR
NOESY
TOCSY
DQF-COSY
Peak Assignment
Distance
Constraint
Dihedral angle
Constraint
Structural Simulation

8. Peak Assignment Strategies
Stage 1 Spin System Identification
Stage 2 Sequence-Specific Assignment

9. In the COSY experiment, magnetization is transferred by scalar coupling. Protons that are more than three chemical bonds apart give no cross signal because the 4J coupling constants are close to 0. Only signals of protons which are two or three bonds apart are visible in a COSY spectrum (red signals). The cross signals between HN and Halpha protons are of special importance because the phi torsion angle of the protein backbone can be derived from the 3J coupling constant between them
The TOCSY experiment correlates all protons of a spin system. Therefore, not only the red signals are visible (which also appear in a COSY spectrum) but also additional signals (green) which originate from the interaction of all protons of a spin system that are not directly connected via three chemical bonds.
Thus a characteristic pattern of signals results for each amino acid from which the amino acid can be identified

11. approximate chemical shifts for various groups in a protein
“random coil” means “not having any specific structure” e.g. helix, sheet
measured in unstructured tetrapeptides
notice that none of these values is above 8.8 (except Trp sidechain NH) or below 0.9
all the amides come between 8 and 8.75
alphas between 4 and 4.8
most methyls between 0.9 and 1.4
some amino acids have identical spin systems and therefore identical signal patterns. They are: cysteine, aspartic acid, phenylalanine, histidine, asparagine, tryptophane and tyrosine ('AMX systems') on the one hand and glutamic acid, glutamine and methionine ('AM(PT)X systems') on the other hand.

13. Hd Ha He Hb Hg Hd Hg Hb He Ha

14. Sequence-Specific Assignment

15. the technique of making the spin-system assignments, followed by sequence-specific assignment using unique fragments of sequence, is known as sequential assignment (Wuthrich)

16. main-chain directed assignment (Englander)
main-chain directed assignment (Englander). This technique does not focus on assigning all the spin systems first. Rather, it focuses on the backbone and links sizable stretches of backbone residues via sequential (i,i+1) nOe’s and other nOe’s that are characteristic of secondary structures. This technique is particularly useful when there is some knowledge of secondary structure beforehand.

18. Chemical Shift & Protein Secondary Structure
For helical conformation
NH & a-H move upfield from the r.c. value
For b-strand conformation
NH & a-H move downfield from the r.c. value
Wishart, Sykes & Richards, J. Mol. Biol. 1991, 222,

19. C.S. difference = d測量－d無序纏捲
化學位移差值(Chemical Shift Index;CSI)
用來判斷蛋白質及胜二級結構之構形傾向，可以顯現與二級結構的關係，計算方法為：
C.S. difference = d測量－d無序纏捲
α-helix
β-sheet NH
upfield
downfield αH βH

20. Temperature Coefficient
溫度每上升1K，backbone NH 質子所感受到周圍環境的不同，所產生的化學位移變化，溫度係數的單位為ppb/K。
溫度係數必須與氫氘交換實驗的結果做比較，才可以判斷背骨胺基質子是否有氫鍵存在。
Baxter & Williamson, J. Biomolecular NMR, 1997, 9 ,

21. gp-41 fusion peptide in SDS micelle, 298 K
Backbone H-D Exchange
2 hour
30 min
gp-41 fusion peptide in SDS micelle, 298 K

22. Backbone H-D Exchange
Amide proton resonance region of the 23-mer fusion peptide of HIV-gp41 in SDS micelle
pH 5
pH 7

23. Nuclear Overhauser Effect

25. Nuclear Overhauser Effect

26. Nuclear Overhauser Effect Brownian motion and NOE

27. Nuclear Overhauser Effect
Distance & NOE Strength
Strong 1.8 ~ 2.7 Å; Medium 1.8 ~ 3.5 Å; Weak 1.8 ~ 5.0 Å

28. 3JNH-aH & dihedral angle Phase sensitive COSY

29. 3JNH-aH & dihedral angle DQF-COSY

30. 3JNH-aH & dihedral angle