Post on 12-Jun-2020
1
Heteronuclear NMR
K.V.R. Charychary@tifr.res.in
Workshop on “NMR and it’s applications in Biological Systems”TIFR November 24, 2009
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)
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
Evolution of spin operator under a pulse
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)
Evolution of spin operator under a pulse
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
Evolution under Couplings (HJ = 2πJ12I1zI2z)
1Η
y
I1x 2I1yI2z
-2I1yI2z -I1x
X Magnetization :HJ=2πJ12I1zI2z
Angle =2πJ12t
13
Evolution under Couplings (HJ = 2πJ12I1zI2z)
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)
Evolution under Couplings (HJ = 2πJ12I1zI2z)
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
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
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
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
σ5 = 2IYSZ cosΩSt1 - 2IYSX sinΩSt1
σ6 = - IX cosΩSt1 - 2IYSX 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
σ5 = 2IYSZ cosΩSt1 - 2IYSX sinΩSt1
σ6 = - IX cosΩSt1 - 2IYSX sinΩSt1
σ7 = - IZ cosΩSt1 +2IySZ sinΩSt1
σ8 = - IZ cosΩSt1 - IX sinΩSt1
σ9 = - Iy cosΩSt1 - IX sinΩSt1
39
2D HSQC
HSQC