Multidimensional NMR

1
Multidimensional NMR
1D Experiment
90o pulse
Time (t)
FT
Frequency (w)
2D Experiment
90ox pulse
90oy pulse
w1
Time (t2)
FT
Time (t1)
w2
2
A 2D NMR Experiment
90oy pulse
90ox pulse
Time (t2)
Time (t1)
Mixing
Preparation
Evolution
Acquisition
3
Bo
B1 Preparation
z
x
? w1t1
w1 Evolution
y
4
Bo
z
?
x
?
? w1t1
y
5
z
Bo
?
x
Mxy
Detection during t2
y
Signal Mxy cos(w2t2)
Signal Mocos ? cos(w2t2)
Signal Mocos (w1t1) cos(w2t2)
6
Multidimensional NMR
1D Experiment
90o pulse
Time (t)
Signal Mo cos(w2t2)
2D Experiment
90ox pulse
90oy pulse
Time (t2)
Time (t1)
Signal Mocos (w1t1) cos(w2t2)
7
z
Bo
x
? w1t1
w1
y
Signal Mocos (w1t1) cos(w2t2)
t10
Mo cos(w2t2)
t2
w2
t1 ?/3w1
Mo ½ cos(w2t2)
w2
t2

t1 ?/2w1
0
w2
t2

8
Bo
x
? w1t1
w1
y
Signal Mocos (w1t1) cos(w2t2)
t1 ?/2w1
0
w2
t2
t1 2?/3w1
-Mo ½ cos(w2t2)
w2
t2

t1?/w1
Mo cos(w2t2)
w2
t2
9
t10
w2
Intensity
t1 ?/3w1
w2
t1
t1 ?/2w1

w2
t1 2?/3w1
w2
FT
t1?/w1
w2
t1 4?/3w1
w1
w2
t1 3?/2w1

w2
t1 5?/3w1
w2
t1 2?w1
w2
10
Signal Mocos (w1t1) cos(w2t2)
t1
t2
w1
w1 w2
w1
w2
w2
11
COSY (Correlation Spectroscopy) Experiment
90oy pulse
90ox pulse
Time (t2)
Time (t1)
Mixing
C
C
Hb
Ha
3Jab
12
Bo
x
? w1t1
w1
y
Signal Mocos (w1t1) cos(w2t2)
C
C
Hb
Ha
Mocos (w1at1) cos(w2at2)
?
Mocos (w1at1) cos(w2bt2)
Signal
Mocos (w1bt1) cos(w2at2)
Mocos (w1bt1) cos(w2bt2)
13
C
C
Hb
Ha
COSY Spectrum
w1
Mocos (w1at1) cos(w2at2)
wb
Mocos (w1at1) cos(w2bt2)
Mocos (w1bt1) cos(w2at2)
wa
Mocos (w1bt1) cos(w2bt2)
w2
wb
wa
w2
14
C
C
Hb
Ha
COSY Spectrum
w1
3Jab
Mocos (w1at1) cos(w2at2)
wb
3Jab
Mocos (w1at1) cos(w2bt2)
Mocos (w1bt1) cos(w2at2)
wa
Mocos (w1bt1) cos(w2bt2)
w2
wb
wa
3Jab
w2
15
C
C
C
Hb
Ha
Hc
w1
wc
wb
wa
w2
wb
wc
wa
w2
16
Ha
O
3JNa
Alanine
N
C
C
3Jab
HN
C-H3b
w1
w2
wHa
wHb
wHN
w2
17
TOCSY (Total Correlation Spectroscopy) Experiment
Spin-lock pulse
90oy pulse
Time (t2)
Time (t1)
Mixing
Ha
O
N
C
C
HN
C-H3b
Coupling between all protons in amino acid (spin
system)
18
Ha
O
3JNa
Alanine
N
C
C
3Jab
HN
C-H3b
w1
w2
wHa
wHb
wHN
w2
19
(No Transcript)
20
NOESY (NOE Spectroscopy) Experiment
90oy pulse
90ox pulse
Time (t2)
Mixing time (t)
Time (t1)
Through-Space Coupling
21
observable if lt 4Å
strength of coupling 1/r6
w1
wb
integrated intensity (r)
wa
w2
wb
wa
22
Can combine NOESY and COSY/TOCSY to assign
peptides
Ha3
Ha2
O
O
N
C
N
C
C
C
HN3
HN2
R3
R2
4.0 ppm
NOESY
wa
wa
wa
5.0 ppm
wN
wN
wN
9.0 ppm
8.0 ppm
23
Can combine NOESY and COSY/TOCSY to assign
peptides
Ha3
Ha2
O
O
N
C
N
C
C
C
HN3
HN2
R3
R2
4.0 ppm
COSY
wa2
wa3
wa1
5.0 ppm
wN1
wN3
wN2
9.0 ppm
8.0 ppm
w2
24
Can combine NOESY and COSY/TOCSY to assign
peptides
Ha3
Ha2
O
O
N
C
N
C
C
C
HN3
HN2
R3
R2
4.0 ppm
NOESY
wa2
wa3
wa1
5.0 ppm
wN1
wN3
wN2
9.0 ppm
8.0 ppm
25
Can combine NOESY and COSY/TOCSY to assign
peptides
Ha3
Ha2
O
O
N
C
N
C
C
C
HN3
HN2
R3
R2
4.0 ppm
NOESY
wa2
wa3
wa1
5.0 ppm
wN1
wN3
wN2
9.0 ppm
8.0 ppm
26
Problem Set NOESY
wHa
wHN
27
Common Observed NOEs
Hai2
Hai1
O
O
N
C
N
C
C
C
HNi2
HNi1
Ri2
Ri1
Intraresidue
aN (i,i)
aR (i,i)
Sequential
aN (i,i1)
aR (i,i1)
NN (i,i1)
28
Some Common Observed NOEs
Hai2
Hai1
O
O
N
C
N
C
C
C
HNi2
HNi1
Ri2
Ri1
Medium Range
Intraresidue
aN (i,i2)
aN (i,i)
aN (i,i3)
aR (i,i)
aN (i,i4)
Sequential
Long Range
aN (i,i1)
HH (i,igt4)
aR (i,i1)
NN (i,i1)
H
H
29
Some Common Observed NOEs
30
Assigning Secondary Structure from Common
Observed NOEs
31
Assigning Secondary Structure from Common
Observed NOEs
32
Heteronuclear multidimensional spectroscopy
Time (t1)
N15
1JHN
Time (t2)
HN
wN
wN
wH
wH
33
Heteronuclear Single Quantum Coherence (HSQC)
experiment
90o
180o
90o
180o
90o
180o
Time (t2)
1H pulse (B1wH/gH)
15N pulse (B1wN/gN)
Time (t1)
Acquisition
Mixing
Evolution
Preparation
N15
HN
34
t10
wH
Intensity
t1 ?/3wN
wH
t1
t1 ?/2wN

wH
t1 2?/3wN
wH
FT
t1?/wN
wH
t1 4?/3wN
wN
wH
t1 3?/2wN

wH
t1 5?/3wN
wH
t1 2?wN
wH
35
HSQC 100 amino acid protein
15N (ppm)
1H (ppm)
Why no prolines?
36
Unfolded Protein
Folded Protein
E2F1 CC-MB
15N
15N (ppm)
15N (ppm)
1H (ppm)
1H (ppm)
37
HSQC of Aggregated Protein
38
3-D Backbone Correlation Spectroscopy
1JNC
O
Time (t1)
N15
C13
1JHN
Time (t2)
HN
Time (t3)
HNCO
wN
wC
wH
39
t10
wH
Intensity
t1 ?/3wc
wH
t2
t1 ?/2wc

wH
t1 2?/3wc
wH
FT
t1?/wc
wH
t1 4?/3wc
wc
wH
t1 3?/2wc

wH
t1 5?/3wc
wH
t1 2?wc
wH
40
t10
Intensity
t1 ?/3wN
wC
wH
t1
t1 ?/2wN
FT
t1 2?/3wN
wC
wH
wN
t1?/wN
wC
t1 4?/3wN
wH
41
HNCO Pulse Sequence
42
(No Transcript)
43
Chemical Exchange
kex
H
H
Exchange rate
B
A
wHA ? wHB
kex
H
H
Exchange rate
B
A
44
Chemical Exchange
kex
H
H
Exchange rate
B
A
kex
wHB
wHA
45
Chemical Exchange
kex
wHB
wHA
kex ltlt Dw
wHA
wHB
kex Dw
wH
kex gtgt Dw
ltwHgt
46
Ligand Titration-Chemical Shift Mapping
H
H
H
H
Slow Exchange
Intensity population
Unbound
Saturated
wN
wN
wH
wH
Protein HSQC
47
Ligand Titration-Chemical Shift Mapping
H
H
H
H
Fast Exchange
wN
w wuUwBB
UB

wH
Protein HSQC
48
Ligand Titration-Chemical Shift Mapping
49
Ligand Titration-Chemical Shift Mapping
50
Amide Proton Exchange
O
O
H
N
N
C
C
HN
Amide protons will exchange with solvent H2O
O
O
D2O
N
N
C
C
HN
D
Is this slow or fast exchange?
51
Amide Proton Exchange
O
O
H
N
N
C
C
HN
Amide protons will exchange with solvent H2O
5
3
0.1 min-1
log k (min-1)
1
10 min-1
-1
1
3
5
7
pH
52
Exchangable Proton Protection
D2O
kexA ltlt kexB
wN
wN
wH
wH
53
Protein Folding and Protection Factors
H
H
Folded/Native
Unfolded/Denatured
Protection Factor (P) kex(U) / kex(N)
Experimentally how are these rates measured?