陳金榜 (Chinpan Chen)

Room #: N133

Tel.: 2652-3035

(I) NMR theory and experiments

(II) Structure determination: Protein

(III) Structure determination: Nucleic acid

Dr. Yuan-Chau Lou IBMS, Academia Sinica

Dr. Iren Wang IBMS, Academia Sinica

outline 3 NMR experiments

4

Conclusion5

NMR parameters2

NMR principles 1

NMR resonance assignment

NMR principles

Nuclear spin (I) : A = Z + N

The number of neutrons

The number of protons(atomic number)

Nominal atomic mass

NMR can’t be detected when nuclei with I ＝ 0 .

NMR can be detected when nuclei with I≠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 charge Z, 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 N odd) will exhibit an integer value of I. [2H(I=1), 14N(I=1) and 10B(I=3)].

A nucleus with odd mass (Z odd and 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)].

NMR principles

Number of spin states (2I+1) :

0 △E

E

B0

m=-1/2

m=+1/2

E=-mB0(γh/2π)

B0: magnetic field strength h : Planck’s constant m: spin quantum number γ: magnetogyric ratio

A nucleus with spin I can 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.

Properties of nuclei in NMR studies

Isotope

1H

2H

3H

12C

13C

14N

15N

16O

17O

19F

31P

Spin

1/2

1

1/2

0

1/2

1

1/2

0

5/2

1/2

1/2

Frequency (MHz) at 11.74T

500.0

76.7

533.3

-------

125.7

36.1

50.7

------

67.8

470.4

202.4

Nature abundance ( ％ )

99.98

1.5×10-2

0

98.89

1.108

99.63

0.37

~ 100

3.7×10-2

100

100

Relativesensitivity

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

X-RAY ULTRAVIOLET INFRAREDMICRO-WAVE

RADIOFREQU-ENCY

Energylowhigh Frequency (ν)

ULTRAVIOLET VISIBLEVIBRATIONAL

INTRARED

NUCLEARMAGNETIC

RESONANCE

5 m1 m15μ2.5μ

200 nm 400 nm 800 nm

Wavelength (λ)short long

A Portion of the Electromagnetic Spectrum Showing the Relationship of the Vibrational Infrared to Other Types of Radiation.

Region of Spectrum

X-rays

Ultraviolet/Visible

Infrared

Microwave

Radiofrequencies

Energy Transitions

Bond Breaking

Electronic

Vibrational

Rotational

Nuclear Spin ( Nuclear Magnetic Resonance ) Electron Spin ( Electron Spin Resonance )

Types of Energy Transitions in Each Region of the Electromagnetic Spectrum

Conceptual block diagram of the pulsed fourier transform NMR experiment

Sample

Magnetization

Response

Data (FID)

Spectrum

Storage

Magnet

Perturbation

Detection

Fourier transformation

The normal detection pulse sequence

FID

Data acquisition ( detection )

pulse

delay

Z

M

Y

XB1

(|| B0) Z

M1

Y

X

M2

1. 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.

60 10/

ppm

referencesignal

8 6 4 2 0 -2

CH3

H

H

H

CH3 CH4

(CH3)4Si

CH3Li

Chemical shift δ(ppm)downfield high

frequencyupfield low frequency

Increased shielding

Increased deshielding

Diamagnetic Anisotropy in Benzene

Anisotropy Caused by the Presence of π Electrons in Some Common

Multiple Bond Systems

COMPOUND CH3X CH3F CH3OH CH3Cl CH3Br CH3I CH4 (CH3)4Si

ELEMENT X F O Cl Br I H Si

ELECTRONEGATIVITY OF 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

The Dependence of the Chemical Shift of CH3X on the Element X

N129

Backbone NH

Aromatic protons

Indo NH of Trp

H2O

H H

Other side-chain protons

Methyl protons

Groups of Hydrogen Atoms in the Common Amino Acid Residues with similar Random Coil 1H Chemical Shiftsa.

10 5 1 δ(ppm)

2H(H) Ring α,β(S.T) CH3

β(a)β(b)NH(W)* NH(bb)* NH(sc)*

CodeCH3

β(a)β(b)

α,β(S.T)Ring2H(H)

NH(sc)*NH(bb)*NH(W)*

δ(ppm)0.9-1.41.6-2.32.7-3.31.2-3.33.9-4.86.5-7.77.7-8.6

6.6-7.68.1-8.810.2

Comments

βH of V, I, L, E, Q, M, P, R, KβH of C, D, N, F, Y, H, WOther aliphatic CHAll αH, βH of S and TAromatic CH of F, Y, W; 4H of H2H of h in the pH range 1-11

Side chain NH of N, Q, K, RBackbone NHIndole NH of W

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

2. 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 ).

J J J J J

(Quartet) δCH2

(Triplet) δCH3

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

pascal’s Triangle

ψ Ψ

Cα

Cβ

Cγ

ω

O

N

χ1

χ2

C’

N

Dihedral Angles: ψ, Ψ, ω, χ1, χ2

Parameters for regular polypeptide conformations

ββpα-helix310-helix

π-helixPolyproline IPolyproline II

Polyglycine II

ψ-139-119-57-49-57-83-78-80

Ψ+135+113-47-26-70+158+149+150

ω-1781801801801800180180

Residuesper turn

2.02.03.63.04.43.333.03.0

Translationper residue

3.43.21.502.001.151.93.123.1

Bond Angle (deg)

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).

Karplus Equations:

8.1cos6.1cos5.9

8.1)120cos(6.1)120(cos5.9

9.1)60cos(4.1)60(cos4.6

112

23

112

13

23

J

J

JNH

Dihedral Angles: , ψ, ω, χ1, χ2

3. 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.

obs

irr

r

r

2

1

Example: the maximum NOE signal enhancement of a13C{1H} signal.

988.11026.67

105.267

2

1

2

16

6

obs

irr

r

r

NOEαR-6 (R: interproton distance)

ii+1

i+2

ParameterdαN(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-helix2.63.54.43.44.22.84.22.5-4.12.5-4.1

( 4)≦slow

310-helix2.63.43.83.3(>4.5)2.64.12.9-4.43.1-5.1

( 4)≦slow

β2.82.2

4.3

3.2-4.5

2.33.23.3( 9)≧slow

βP

2.82.2

4.2

3.7-4.7

4.83.04.0( 9)≧slow

Short sequential and medium-range 1H-1H distances, vicinalcoupling constants, and amide hydrogen exchange rates.

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

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)

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

4444444 4444444 9999999

The thickness of the lines is an indication of the intensity of the NOEs

The values of J coupling are approximate.

4. 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.

5. 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

2

1

Tlinewidth

NMR parameters

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,

C N C C

O H H O

R

＋ D2O C N C C

O D H O

RExchange rate study of amide proton can be used to identify the amide protons that form H-bond or are shielded from the solvent.

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

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

Backbone NH

Aromatic protons

Indo NH of Trp

H2O

H H

Other side-chain protons

Methyl protons

Resonance assignment strategiesfor 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 resolvingoverlapping NMR resonances.

PREPARATION EVOLUTION MIXING DETECTION

t1

t1

t1t2

t2

t2

τM

(A)

(B)

(C)

(D)

(A) The elements of a generalized two- dimensional NMR experiment

(B) DQFCOSY experiment

(C) NOESY experiment

(D) TOCSY experiment

Cα

Hα3

Gly

NC

OH

3.97

8.39COSY

Hα2

NH

H (p

pm)

H (ppm)

TOCSY

Hα

NH

H (p

pm)

45.0

H (ppm)

173.6

Hα2

Hα3

Hα3

108.9

Cα

HαCβ

Hβ

Ala

N C

OH

1.39

4.35

8.25

COSY

Hα

Hβ

NH

H (p

pm)

H (ppm)

TOCSY

Hα

Hβ

NH

H (p

pm)

52.5

H (ppm)

19.0

177.1

Hβ

Hβ

122.5

Cα

Hα

Cβ

Hβ

Val

N C

OH

0.97

4.18

8.44

COSY

Hα

Hβ

NH

H (p

pm)

H (ppm)63.0 177.1

Cγ2 Cγ1

Hγ2

Hγ2 Hγ2

Hγ1

Hγ1Hγ1

2.13

0.94

31.7

Hγ1Hγ2

TOCSY

Hα

Hβ

NH

H (p

pm)

H (ppm)

Hγ1Hγ2

121.1

Cα

Hα

Cβ

Hβ3

Ser

NC

OH 8.38

COSY

Hα

NH

H (p

pm)

H (ppm)

4.50

173.7

3.88

3.88

58.3

Hβ2

62.7

Hβ2

Hβ3

Oγ

Hγ

TOCSY

Hα

NH

H (p

pm)

H (ppm)

Hβ2

Hβ3

116.7

C C

OH

R1

NH3 N

H

C C

OH

R2

N

H

C C

OH

R3

N

H

C C

OH

R4

N

H

C C

OH

R5

N

H

C C

OH

R6

F V Q A T A’

+

α-helical NOEs dαN(i,i+3), dNN(i,i+1)

Val

Gln

Ala

Ala’

Thr

Fingerprint region

Amide protons (ppm)

Hα (

ppm

)

NOESY

Phe

Hα

2D 1H NMR Resonance Assignments

(1) 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 CH2 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

Methods for resolving overlappingNMR 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).

NMR parameters

α

β

13C 15N OH

α

β

β

140 Hz

30 to 40 Hz

55 Hz

90 to 100 Hz

15 Hz11 Hz

用於多維異核核磁共振的單鍵異核偶合常數之概要圖

130.0

125.0

120.0

115.0

110.0

105.0

ppm

10.0 9.0 8.0 7.0 6.0 ppm

HSQC

1H Chemical Shifts

15N

Ch

em

ical

Sh

ifts

Triple (1H, 13C, and 15N) Resonance Experiments

Through-bond experiments:

(a) 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

NMR experiment

二項常用的三核共振實驗之脈衝圖譜

24 25 26F3

F1

sequence

Data reduction for 3D NMR. Once the 15N and 1H frequencies Of each amide group have been tabulated, strips running parallel to F1 can be extracted by software in order to build a 2 spectrum. The strips are order arbitrarily before assignment and according the sequence after assignment (D. Marion).

NMR Resonance Assignments Using Triple Resonance Experiments

(1) 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, C of Thr, Ile, Val, and Pro (> 60 ppm); C of Thr, and Ser (> 60 ppm) b. C of Ala (~ 50 ppm), C of Gly (~ 45ppm), and C of Pro (~ 50 ppm) c. C of Leu, Asp, Asn, Ile, Phe, and Tyr (36~ 43ppm); C 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.

F6

122.24122.24

Q7

116.61116.61

K8

117.55117.55

K9

115.36115.36

H10

106.93106.93

L11

120.05120.05

T12

113.80113.80

D13

124.43124.43

T14

113.80113.80

K15

124.43124.43

70.00

60.00

50.00

40.00

30.00

20.00

10.00

CBCACONH HNCACB

8.81 8.13 7.46 8.27 7.83 8.32 8.25 8.28 8.12 8.24

70.00

60.00

50.00

40.00

30.00

20.00

10.00F6 Q7 K8 K9 H10 L11 T12 D13 T14 K15

122.24122.24 116.61116.61 117.55117.55 115.36115.36 106.93106.93 120.05120.05 113.80113.80 124.43124.43 113.80113.80 124.43124.43 D2 /ppmD2 /ppm

8.81 8.13 7.46 8.27 7.83 8.32 8.25 8.28 8.12 8.24

180.000

177.500

175.000

172.500

170.000

180.000

177.500

175.000

172.500

170.000

F6 Q7 K8 K9 H10 L11 T12 D13 T14 K15 F6 Q7 K8 K9 H10 L11 T12 D13 T14 K15

122.24 116.61 117.55 115.36 106.93 120.05 113.80 124.43 113.80 124.43 122.24 116.61 117.55 115.36 106.93 120.05 113.80 124.43 113.80 124.43D1 /ppm

HNCO HNCACOD1 /ppm

8.81 8.13 7.46 8.27 7.83 8.32 8.25 8.28 8.12 8.24 8.81 8.13 7.46 8.27 7.83 8.32 8.25 8.28 8.12 8.24

70.00

60.00

50.00

40.00

30.00

20.00

10.00

5.00

4.00

3.00

2.00

1.00

0.00

F6 Q7 K8 K9 H10 L11 T12 D13 T14 K15

122.24 116.61 117.55 115.36 106.93 120.05 113.80 124.43 113.80 124.43

F6 Q7 K8 K9 H10 L11 T12 D13 T14 K15

122.24 116.61 117.55 115.36 106.93 120.05 113.80 124.43 113.80 124.43D1 /ppm D1 /ppm

HCCONH CCONH

8.81 8.13 7.46 8.27 7.83 8.32 8.25 8.28 8.12 8.24 8.81 8.13 7.46 8.27 7.83 8.32 8.25 8.28 8.12 8.24

10.00

7.50

5.00

2.50

0.00

F6 Q7 K8 K9 H10 L11 T12 D13 T14 K15

122.24 116.61 117.55 115.36 106.93 120.05 113.80 124.43 113.80 124.43D1 /ppm

TOCSY-HSQC

F6 Q7 K8 K9 H10 L11 T12 D13 T14 K15

122.24 116.61 117.55 115.36 106.93 120.05 113.80 124.43 113.80 124.43D1 /ppm

NOESY-HSQC

8.81 8.13 7.46 8.27 7.83 8.32 8.25 8.28 8.12 8.24 8.81 8.13 7.46 8.27 7.83 8.32 8.25 8.28 8.12 8.24

10.00

7.50

5.00

2.50

0.00

130.0

125.0

120.0

115.0

110.0

105.0

ppm

10.0 9.0 8.0 7.0 6.0 ppm

HSQC

D2

W3

E4

T5

F6

Q7

K8

K9

H10

L11

T12

D13

T14

K15

K16

V17

K18

C19

D20

V21

E22

M23

A24

K25

A26

L27

K32

T33

N34

T35

N92

I37

Y38A39

L40

G42

R43

V44

K45

A46

L47

C48

K49

N50

I51

R52

D53

N54

T55

D56

V57

L58

S59

R60

D61

A62

F63

S80

S81

T83

N84

T85

I86

C87

I88

T89

C90

V91

A99

G100

V101

G102

C104

C75

H76

Y77

K78

L79

Q93

L94

L64

L65

F28

D29

C30I96

H97

F98

F36R70

Q67L73

K73

I71

W3(N1H)

S103

Q7*

N84*

N92*

N54*N50*

Q93*

N34*

Q67*

R*R*R*

R*C68

Three-dimensional structure determination by simulated annealing using X-PLOR ( CNS ) program

tornoerepimproperanglebondtotal EEEEEEE +++++=

Keep the correctness of protein geometry

The energy terms of experimental data

K82K11

K67

K68

K32

K30

D85

D79

D17

E14

E76 K19K77

K63D42

E36E35

D33

D57

K60

K78

180-0.3 0.3

(C)

C-

N-

(A)

β1

β2β3

β4

β5

β6β8

β7 β9

β10

Loop I

Loop II

Loop IV

Loop V

Loop III

(B)

(A) Superimposition of backbone atoms for 20 porcine MSP structures (B) The

ribbon and (C) surface representations of the averaged structure of porcine MSP

(A) (B)

(C)

The flowchart of the protein structure determination from NMR data.

Structure refinement using Molecular dynamics simulation

Calculation of initial structureusing distance geometry

Extraction of Structuralinformation

Sequence-specificResonance assignment

NMR spectroscopy1D, 2D, 3D, …

Protein in solution~0.5 ml, 2 mM concentration Sample preparation:

protein isolation

purification,

characterization,

cloning,

isotopic labelling.

Sample preparation:

protein isolation

purification,

characterization,

cloning,

isotopic labelling.

Secondary

structure of

protein

Secondary

structure of

protein

Distances between

protons (NOE),

Dihedral angles(J

coupling), Amide-

proton exchange,

Chemical shifts

index

Distances between

protons (NOE),

Dihedral angles(J

coupling), Amide-

proton exchange,

Chemical shifts

index

Final 3DstructuresFinal 3D

structures

High-throughput NMR structure determinationHigh-throughput NMR structure determination

1. Cloning, protein expression and purification: 15N/13C- and/or 2H/13C/15N-labeled protein; specifically amino acid-labeled protein.

2. NMR hardwares: Using higher-field NMR instrument (800 MHz or 900 MHz) and Cryoprobe.

3. NMR experiments: performing RDC and TROSY exp.

3. Data analysis: Using automatic resonance assignment program (Autoassign, Ansig, etc.)

4. Structure calculation: Aria, Cyana, etc.