Presentazione di PowerPointiupab/WORKSHOP_KVR_LECTURE4_2009.pdf · 1 Heteronuclear NMR K.V.R. Chary [email protected] Workshop on “NMR and it’s applications in Biological Systems”

1

Heteronuclear NMR

K.V.R. [email protected]

Workshop on “NMR and it’s applications in Biological Systems”TIFR November 24, 2009

mailto:[email protected]

2

NATIONAL FACILITY FOR HIGH FIELD NMRIUPAB sponsored International Workshop on

NMR and its applications in Biological SystemsNovember 23-30, 2009; Venue: Auditorium

Time Monday (23/11) Tuesday(24/11) Wednesday(25/11) Thursday(26/11) Friday(27/11)

09:30-10:30 9:00 Registration9:30 Welcome

Biomolecular NMR (KVRC)

ELEPHANTACAVES

Advanced MD NMR-II (KVRC)

NMR in Drug Design (VR)

10:30-11:00 TEA BREAK

11:00-12:00 An Overview (RVH) MagnetizationTransfer (PKM)

ELEPHANTACAVES

NMR of Nucleic Acids (KVRC)

MRI-I(GG)

12:00-13:00 NMR parameters (PKM)

2D COSY & NOESY (RVH)

ELEPHANTACAVES

Membranes(SS)

Solid-State NMR (PKM)

13:00-14:00 LUNCH BREAK

14:00-15:00 Pulsed NMR (RVH)

Applications of 2D NMR (MVJ)

Heteronuclear NMR (KVRC)

Metabonomics & Cell NMR(HMS)

NMR of Large Systems (RVH)

15:00-16:00 Echoes(KVRC)

Advanced MD NMR-I (KVRC)

Labeling Schemes (VR)

NMR of Proteins (RVH)

MRI-II(GG)

16:00-16:30 TEA BREAK

16:30-17:30 NMR Hardware(MVN/AG69)

Tutorials/ AG69(SS&MVJ)

Tutorials/ AG69(SS & MVJ)



Saturday(28/11) FREE

Sunday (29/11) Laboratory Demonstration at National Facility for High Field NMR (MVJ)

Monday (30/11) Laboratory Demonstration at National Facility for High Field NMR (MVJ)

3

RECAP

4

[I1x, I1y] = iI1z

[I1y, I1z] = iI1x

[I1z, I1x] = iI1y

[I2x, I2y] = iI2z

[I2y, I2z] = iI2x

[I2z, I2x] = iI2y

5

Evolution of spin operator under a pulse

1Η

X

Iz -Iy

Iy -Iz

(π/2)X Pulse:

6


1Η

Y

Iz Ix

-Ix -Iz

(π/2)Y Pulse:

7

• Iz→ Iz cos(β) + Ix sin(β)

• Iz→ Iz cos(β) − Iy sin(β)


βy

βx

8

Y

X

Y

X

Ωtt1

My Sin (Ωt)

Mx Cos (Ωt) Mx

Evolution under Chemical Shift (Hδ = ΩΗIz)

9

Rotation about the z-axis• I1x→ I1x cos(Ωt) + I1y sin(Ωt)

• I1y→ I1y cos(Ωt) − I1x sin(Ωt)

• I1z→ I1z

Evolution under Chemical Shift (Hδ = ΩΗIz)

10

Evolution under Chemical Shift (Hδ = ΩΙIz)

1Η

y

Ix Iy

-Iy -Ix

X/Y Magnetization :

Hδ= ΩΙIz

Angle = ΩΙ t

11

• I1x→ I1x cos(πJ12t) + 2I1yI2z sin(πJ12t)

• I1y→ I1y cos(πJ12t) − 2I1xI2z sin(πJ12t)

• 2I1xI2z → 2I1xI2z cos(πJ12t) + I1y sin(πJ12t)

• 2I1yI2z → 2I1yI2z cos(πJ12t) − I1x sin(πJ12t)

Evolution under Couplings (HJ = 2πJ12I1zI2z)

12


1Η

y

I1x 2I1yI2z

-2I1yI2z -I1x

X Magnetization :HJ=2πJ12I1zI2z

Angle =2πJ12t

13


1Η

x

I1y 2I1xI2z

-2I1xI2z -I1y

Y Magnetization :HJ=2πJ12I1zI2z

Angle =2πJ12t

14

• I1x→ I1x cos(πJ12t) + 2I1yI2z sin(πJ12t)

• I1y→ I1y cos(πJ12t) − 2I1xI2z sin(πJ12t)

• 2I1xI2z → 2I1xI2z cos(πJ12t) + I1y sin(πJ12t)

• 2I1yI2z → 2I1yI2z cos(πJ12t) − I1x sin(πJ12t)


15

In-phase/anti-phase magnetization

Ikx Ilx

Ω k Ω l

Iky Ily

Ω k Ω l

α l β l α k β k

α l β l α k β k

2IkxIlz 2IkzIlx 2IkyIlz 2IkzIly

Ω k Ω l Ω k Ω l

α l β l α k β k

α l β l α k β k

Ikx Ilx

Ω k Ω l

Iky Ily

Ω k Ω l

α l β l α k β k

α l β l α k β k

Ikx Ilx

Ω k Ω l

Iky Ily

Ω k Ω l

Ikx Ilx

Ω k Ω l

Iky Ily

Ω k Ω l

α l β l α k β k

α l β l α k β k

α l β l α k β k

α l β l α k β k


Ω k Ω l Ω k Ω l

α l β l α k β k

α l β l α k β k


Ω k Ω l Ω k Ω l

α l β l α k β k

α l β l α k β k

16

For large biomolecules (Mr > 10 kDa)

Overcrowding of peaks.Decrease in sensitivity due to short T2

values.1H based approaches fail.

Hence there was a need for an alternative strategy.

17

Other alternatives• Use of NMR active nuclei such as 13C and 15N

which have:Large coupling with 1H (90-140 Hz)Large dispersion in chemical shifts

• But these nuclei have low natural abundance 13C = 1 : 10015N = 1 : 300

Need for isotope labeling

18

Bio-synthetic over-expressionof the protein

19

Isotope labeling ………The host micro-organism, overexpressing the protein of interest, is grown on minimal media containing [13C6] glucose as the sole source of carbon or/and [15N] labeled ammonium chloride as the sole source of nitrogen.

Fractionally or Uniformly !!!

20

15N labeling results in ……

Record 2D [15N-1H] HSQC experiment. No. of Peaks = No. of amide 15N-1H pairs

~ No. of amino acid residues present in the protein

Experimental time ~ 10 mins (with 1mM sample)

1HN 1Hα

15N 12Cα

R

O 1HN 1Hα

12C’ 15N 12Cα

R’

12C’

O

21

2D 15N-1H HSQC

2D HSQC

22

Double resonance 3D NMR spectrum

15N edited NOESY spectrumGlutaredoxin85 amino acids10 kDa

3D HSQC-NOESY

23

O H H z

H H

N

H

C C

C

C

55 Hz 15 Hz 9 - 11 Hz

4-7 Hz

35 Hz 90 Hz140 Hz

Coupling constants in a doubly labeled (13C & 15N) Protein

24

Triple resonance NMR experiments

• These correlate backbone 1HN, 15N, 1Hα, 13Cα and 13C’ spins and side chain 13Cβ and 1Hα spins using one-bond and two-bond heteronuclear scalar coupling interactions.

• These experiments constitute an alternative to the classical sequential resonance assignment strategy.

25

Triple resonance 3D NMR spectrum

Sahu et al, J. Biomol. NMR., 14, 93-94, 1999.

26

INEPT

Insensitive Nuclei Enhanced by Polarization Transfer

The aim of this experiment is to transfer proton magnetization to a coupled nucleus, such as carbon–13 or nitrogen–15 .

γH = 4 γC & 10 γN

And so the proton magnetization is correspondingly larger. By transferring this magnetization to the insensitive nucleus (13C or 15N), the detected signal from such nucleus will be stronger.

27

x x y

I x x

S

σ0 = γI IZ

σ1 = - γI IY

σ2 = - γI 2 IX SZ

σ3 = - 2 γI IZ SY

σ3 = γI (- 2 IZ SY)

INEPT

0 1 2 3

28

In contrast, if a simple 90x pulse had been applied to equilibrium S spin magnetization(γS SZ) the result would be

Thus,

INEPT Observation

Direct Observation=

γI

γS

(π/2)x

γS SZ γS ( - SY )(π/2)x

INEPT

S

29

The advantage of INEPT observation becomes greater and greater the lower the γ of the S spin.

A further advantage is that the repetition rate of the experiment is set by the relaxation times of the high γ spins, which are typically much shorter than those of the low γ spins.

INEPT

30

x x y

0 1

2

x y

INEPT

I

S

σ0 = γI IZ

σ1 = - γI IY

σ2 = - γI 2 IX SZ

σ3 = + 2 γI IZ SX

σ3 = γI ( IZ SX)

3

(π/2)y Pulse on ‘S’ spin creates additionally γc Sx

Thus, σeff = γI ( IZ SX) + γc Sx

31

HSQCHeteronuclear single–quantum correlation spectroscopy

1H ∆ ∆ ∆ ∆

∆ ∆ ∆ ∆ DECX t1

t2

X y y

Ø2Ø1

0 1 2 3 4 5

σ0 = IZ ; σ1 = - IY ; σ2 = - 2IX SZ ; σ3 = -2IZ SY

σ4 = -2IZ SXCosΩSt1 -2IZSY SinΩSt1

180o I pulse in the middle of t1 decouples the I and S SpinsIZ does not evolve under homonuclear coupling

ΩS is the larmor frequency of the spin S in the rotating frame

32

HSQC

σ4 = 2IZ SY cosΩSt1 - 2IZSX sinΩSt1

σ5 = 2IYSZ cosΩSt1 - 2IYSX sinΩSt1

σ6 = - IX cosΩSt1 - 2IYSX sinΩSt1

33

HMQC

I

DECSt1

0 1

2’ 2 3 4t 2

x x

x φ

Heteronuclear multiple–quantum correlation spectroscopy

σ0 = IZ ; σ1 = - IY ; σ2’ = 2IX SZ ; σ2 = -2IX SY

σ3 = -2IX [SYCosΩSt1 -SX SinΩSt1]

σ4 = 2IXSZCosΩSt1+2IxSX SinΩSt1

Observable Non-observable SQC superposition of

ZQC and 2QC

34

Record 2D [15N-1H] HSQC experiment. No. of Peaks = No. of amide 15N-1H pairs

~ No. of amino acid residues present in the protein

Experimental time ~ 10 mins (with 1mM sample)

1HN 1Hα

15N 13Cα

R

O 1HN 1Hα

13C’ 15N 13Cα

R’

13C’

O

15N labeling results in ……

35

Sensitivity enhanced HSQC

1H ∆ ∆ ∆ ∆

∆ ∆ ∆ ∆ DEC15N t1

t2

x -y -y -y

Ø2Ø1

0 1 2 3 4 5∆ ∆

∆ ∆

Ø2 Ø3 Ø3

Ø5 Ø5 Ø6 Ø6Ø4

Ø1

Sensitivity gain is √2 times compared to HSQC

36

HSQC

σ4 = 2IZ SY cosΩSt1 - 2IZSX sinΩSt1



X

We ignored this term so far !!!

37

Sensitivity enhanced HSQC

1H ∆ ∆ ∆ ∆

∆ ∆ ∆ ∆ DEC15N t1

t2

x -y -y -y

Ø2Ø1

0 1 2 3 4 5 6 7 8 9∆ ∆

∆ ∆

Ø2 Ø3 Ø3

Ø5 Ø5 Ø6 Ø6Ø4

Ø1

38

Sensitivity enhanced HSQCσ4 = 2IZ SY cosΩSt1 - 2IZSX sinΩSt1



σ7 = - IZ cosΩSt1 +2IySZ sinΩSt1

σ8 = - IZ cosΩSt1 - IX sinΩSt1

σ9 = - Iy cosΩSt1 - IX sinΩSt1

39

2D HSQC

HSQC

Documents

Presentazione di PowerPointiupab/WORKSHOP_KVR_LECTURE4_2009.pdf · 1 Heteronuclear NMR K.V.R. Chary [email protected] Workshop on “NMR and it’s applications in Biological Systems”