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陳金榜 (Chinpan Chen)

Nuclear Magnetic Resonance (NMR). 陳金榜 (Chinpan Chen). Room #: N133 Tel.: 2652-3035. 1. NMR p rinciple s. 2. NMR parameters. 3. NMR experiments. 4. 5. Conclusion. outline. NMR resonance assignment. NMR principle s. Nuclear spin (I) :. A = Z + N. Nominal atomic mass.

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陳金榜 (Chinpan Chen)

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  1. Nuclear Magnetic Resonance (NMR) 陳金榜 (Chinpan Chen) Room #: N133 Tel.: 2652-3035

  2. 1 NMR principles 2 NMR parameters 3 NMR experiments 4 5 Conclusion outline NMR resonance assignment

  3. NMR principles Nuclear spin (I) : A = Z + N Nominal atomic mass The number of neutrons The number of protons (atomic number)

  4. NMR can’t be detected when nuclei withI＝0. • NMR can be detected when nuclei withI≠0 • Different isotopes of the same element have different nuclear spins, some of which are detectable by NMR, others of which are not. • A nucleus with an even mass A and even chargeZ, and therefore also an even N, will have a nuclear spin I of zero. (12C, 16O and 18O). • A nucleus with an even mass and odd charge (both Z and Nodd) will exhibit an integer value of I. [2H(I=1), 14N(I=1) and 10B(I=3)]. • A nucleus with odd mass (Z oddand N even, or Z even and N odd) will have nuclear spin with an I value that can be expressed as n/2, where n is an odd integer. [1H(I=1/2), 13C(I=1/2) and 17O(I=5/2)].

  5. NMR principles Number of spin states (2I+1) : m=-1/2 E=-mB0(γh/2π) B0: magnetic field strength h:Planck’s constant m: spin quantum number γ:magnetogyric ratio △E 0 E m=+1/2 B0 A nucleus with spin Ican have 2I+1 spin states. Each of these states has its own spin quantum number m(m=-I, -I+1, ‧‧‧, I-1, I). For nuclei with I=1/2, only two states are possible: m=+1/2 and m=-1/2.

  6. Properties of nuclei in NMR studies Isotope 1H 2H 3H 12C 13C 14N 15N 16O 17O 19F 31P Nature abundance (％) 99.98 1.5×10-2 0 98.89 1.108 99.63 0.37 ~ 100 3.7×10-2 100 100 Relative sensitivity 1.0 9.65×10-3 1.21 ------ 1.59×10-2 1.01×10-3 1.04×10-3 ------- 2.91×10-2 0.83 6.63×10-2 Frequency (MHz) at 11.74T 500.0 76.7 533.3 ------- 125.7 36.1 50.7 ------ 67.8 470.4 202.4 Spin 1/2 1 1/2 0 1/2 1 1/2 0 5/2 1/2 1/2

  7. Frequency (ν) high low Energy X-RAY ULTRAVIOLET INFRARED MICRO- WAVE RADIO FREQU- ENCY ULTRAVIOLET VISIBLE VIBRATIONAL INTRARED NUCLEAR MAGNETIC RESONANCE 2.5μ 15μ 1 m 5 m 200 nm 400 nm 800 nm short Wavelength (λ) long A Portion of the Electromagnetic Spectrum Showing the Relationship of the Vibrational Infrared to Other Types of Radiation.

  8. Types of Energy Transitions in Each Region of the Electromagnetic Spectrum Region of Spectrum Energy Transitions X-rays Ultraviolet/Visible Infrared Microwave Radiofrequencies Bond Breaking Electronic Vibrational Rotational Nuclear Spin ( Nuclear Magnetic Resonance ) Electron Spin ( Electron Spin Resonance )

  9. Conceptual block diagram of the pulsed fourier transform NMR experiment Detection Data (FID) Sample Magnet Fourier transformation Spectrum Magnetization Perturbation Response Storage

  10. Z (|| B0) M Y B1 X The normal detection pulse sequence delay FID pulse Data acquisition ( detection ) Z M1 Y M2 X

  11. Chemical shift (δ)defines the location of a nmr line along the rf axis. It is measured relative to a reference compound. In frequency units the chemical shift is proportional to the applied static magnetic field, and therefore chemical shifts are customarily quoted in parts per million (ppm) units. CH3 H (CH3)4Si CH3Li CH3 CH4 H H 8 6 4 2 0 -2 Chemical shift δ(ppm) downfield high frequency Increased shielding upfield low frequency Increased deshielding

  12. Diamagnetic Anisotropy in Benzene Anisotropy Caused by the Presence of π Electrons in Some Common Multiple Bond Systems

  13. The Dependence of the Chemical Shift of CH3X on the Element X COMPOUND CH3X CH3F CH3OH CH3Cl CH3Br CH3I CH4 (CH3)4Si ELEMENT X F O Cl Br I H Si ELECTRONEGATIVITYOF X 4.0 3.5 3.1 2.8 2.5 2.1 1.8 CHEMICAL δ 4.26 3.40 3.05 2.68 2.16 0.23 0 SHIFT τ 5.74 6.60 6.95 7.32 7.84 9.77 10 N129

  14. Other side-chain protons H2O Aromatic protons Methyl protons Ha Backbone NH Ha Indo NH of Trp

  15. Groups of Hydrogen Atoms in the Common Amino Acid Residues with similar Random Coil 1H Chemical Shiftsa. a In model peptides the labile protons (identified by *) are only observed in H2O solution. The singlet resonance of εCH3 in met is at 2.13 ppm NH(W)* NH(bb)* NH(sc)* β(b) β(a) 2H(H) Ring α,β(S.T) CH3 10 5 1 δ(ppm) Code CH3 β(a) β(b) α,β(S.T) Ring 2H(H) NH(sc)* NH(bb)* NH(W)* δ(ppm) 0.9-1.4 1.6-2.3 2.7-3.3 1.2-3.3 3.9-4.8 6.5-7.7 7.7-8.6 6.6-7.6 8.1-8.8 10.2 Comments βH of V, I, L, E, Q, M, P, R, K βH of C, D, N, F, Y, H, W Other aliphatic CH All αH, βH of S and T Aromatic CH of F, Y, W; 4H of H 2H of h in the pH range 1-11 Side chain NH of N, Q, K, R Backbone NH Indole NH of W

  16. Spin-spin coupling constant (J)characterize scalar interactions (through-bond) between nuclei linked via a small number of covalent bonds in a chemical structure. J is field independent and is customarily quoted in hertz (Hz ). (Triplet) δCH3 (Quartet) δCH2 J J J J J

  17. pascal’s Triangle Singlet Doublet Triplet Quartet Quintet Sextet Septet 1 1 1 1 2 1 1 3 3 1 1 4 6 4 1 1 5 10 10 5 1 1 6 15 20 15 6 1

  18. Cγ χ2 Cβ χ1 N Cα ω ψ Ψ N C’ O Dihedral Angles: ψ, Ψ,ω, χ1, χ2

  19. Parameters for regular polypeptide conformations Residues per turn 2.0 2.0 3.6 3.0 4.4 3.33 3.0 3.0 Translation per residue 3.4 3.2 1.50 2.00 1.15 1.9 3.12 3.1 Bond Angle (deg) β βp α-helix 310-helix π-helix Polyproline I Polyproline II Polyglycine II Ψ +135 +113 -47 -26 -70 +158 +149 +150 ω -178 180 180 180 180 0 180 180 ψ -139 -119 -57 -49 -57 -83 -78 -80 Adapted from G. N. Ramachandran and V. Sasisekharan, Adv. Protein Chem. 23, 283-437(1968); IUPAC-IUB Commission on biochemical Nomemclature, Biochemistry 9, 3471-3479 (1970).

  20. Karplus Equations: Dihedral Angles: ψ, Ψ,ω, χ1, χ2

  21. Nuclear Overhauser enhancement or Nuclear Overhauser effect (NOE)is the fractional change in intensity of one NMR line when another resonance is irradiated in a double irradiation experiment. Nuclear Overhauser effects are due to dipolar interactions (through-space) between different nuclei and are correlated with the inverse sixth power of the internuclear disran. Example: the maximum NOE signal enhancement of a13C{1H} signal. NOEαR-6 (R: interproton distance)

  22. i+2 i i+1

  23. Short sequential and medium-range 1H-1H distances, vicinal coupling constants, and amide hydrogen exchange rates. Parameter dαN(i,i) 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) dαα (i,j) dαN (i,j) dNN (i,j) 3JHNα(HZ) NH exchange rate α-helix 2.6 3.5 4.4 3.4 4.2 2.8 4.2 2.5-4.1 2.5-4.1 (≦4) slow 310-helix 2.6 3.4 3.8 3.3 (>4.5) 2.6 4.1 2.9-4.4 3.1-5.1 (≦4) slow β 2.8 2.2 4.3 3.2-4.5 2.3 3.2 3.3 (≧9) slow βP 2.8 2.2 4.2 3.7-4.7 4.8 3.0 4.0 (≧9) slow • dαα(i,j), dαN (i,j) and dNN (i,j) refer to interstrand distances. • The first four residues in the α-helix and the first three residues in the 310-helix will have fast amide proton exchange rates. • Every second residue in the flanking strand will have slow amide proton exchange rates

  24. The characteristic patterns of short-range NOEs involving amide, alpha, beta protons observed for ideal α-helices, 310-helices and β-strand. α-helix 1234567 310-helix 1234567 β-strand 1234567 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) 4444444 9999999 4444444 • The thickness of the lines is an indication of the intensity of the NOEs • The values of J coupling are approximate.

  25. Longitudinal relaxation time or spin-lattice relaxation time (T1)describes the rate at which the magnetization returns to the thermodynamic equilibrium orientation along the static magnetic field after a rf pulse. • Transverse relaxation time or spin-spin relaxation time (T2) describes the decay rate of the effective magnetization observed in the x,y plane after a rf pulse. Half height

  26. Labile protons: －NH ; －OH ; －SH Most of time, NMR signals of these labile protons can not be seen, due to their fast exchange with H2O. However, O H H O O D H O ＋ D2O C N C C C N C C R R Exchange rate study of amide proton can be used to identify the amide protons that form H-bond or are shielded from the solvent. NMR parameters

  27. Structural information • Interproton distances : • NOE α R-6 • Dihedral angles: • J-coupling and Karplus equations • Chemical Shift Index (CSI): • Chemical shift of 1Hα, 13Cα, 13Cβ, 13C’ • Hydrogen bonding: • Amide proton exchange rates

  28. Recommended atom identifiers for the twenty common amino acids follow the 1969 IUPAC-IUB guidelines.

  29. Other side-chain protons H2O Aromatic protons Methyl protons Ha Backbone NH Ha Indo NH of Trp

  30. Resonance assignment strategies for small proteins • Spin system identification : • DQF-COSY and TOCSY experiments • Sequence-specific assignment: • NOESY experiment • For protein < 10 kDa, 2D homonuclear • experiments may be sufficient for resolving • overlapping NMR resonances.

  31. PREPARATION EVOLUTION MIXING DETECTION (A) t2 t1 (B) t2 t1 τM (C) t2 t1 (D) • The elements of a generalized two- dimensional NMR experiment • DQFCOSY experiment • NOESY experiment • TOCSY experiment

  32. 2D 1H NMR Resonance Assignments • Identify spin-system (amino acid type) using 2D COSY & TOCSY • a. Unique residues: Gly, Ala, Val, Ser, Thr • b. Long-chain residues: Arg, Ile, Leu, Lys • c. Containing CH2 residues: Gln, Glu, Met • d. AMX residues: Asn, Asp, His, Phe, Trp, Tyr, Cys • e. No amide proton residue: Pro • (2) Distinguish aromatic residues • (3) Using residues possessing unique side-chain chemical shifts • (4) Sequential assignments based on NOEs • (5) Assign Cis or Trans conformer of Pro residue

  33. Gly H (ppm) Hα3 Hα2 H O 8.39 NH COSY H (ppm) N C 108.9 173.6 45.0 Cα H (ppm) Hα3 Hα2 Hα3 Hα 3.97 NH TOCSY H (ppm)

  34. Fingerprint region Phe Val Gln Hα (ppm) Hα Ala NOESY Thr Ala’ Amide protons (ppm) H O H H O H H O H H O H H O H H O + NH3 C C N C C N C C N C C N C C N C C R1 R2 R3 R4 R5 R6 F V Q A T A’ α-helical NOEs dαN(i,i+3), dNN(i,i+1)

  35. Methods for resolving overlapping NMR resonances • 2D/3D homonuclear NMR experiments • such as 2D-DQFCOSY, 2D-TOCSY, 2D- • NOESY, 3D-NOESY-TOCSY. • 2D/3D heteronuclear NMR experiments • such as 2D-15N-HSQC, 3D-15N-NOESY- • HSQC and triple-resonance experiments ( • 1H, 13C, 15N).

  36. NMR parameters H 用於多維異核核磁共振的 單鍵異核偶合常數之概要圖 13C 15N O β β β 30 to 40 Hz α 55 Hz 11 Hz 140 Hz 15 Hz α 90 to 100 Hz

  37. 1H Chemical Shifts

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

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