Chem806 Identification of organic and inorganic compounds by advance NMR techniques

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Chem-806Identification of organic and inorganic
compounds by advance NMR techniques

• Tool box
• 2D-NMR Homonuclear
• 2D-NMR Heteronuclear
• 3D-NMR

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NMR of protein
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1H NMR spectra of small protein
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Protein folded and unfolded
Folded
Unfolded
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2D-COSY
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2D-COSY NH/CHa expanded region
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3JHH and dihedral angle
H
H
H
H
Gauche and trans conformation can be
distinguished by measuring coupling
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2D-NOESY
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sequential assignment using NOESY

• In the a-helix the neighbouring HN are 2.8
  Ångström apart
• In the ß-strand the distance from Ha in residue
  (i) to HN in residue (i1) is only 2.2 Ångström.

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Schematic presentation NOESY spectrum in the NH
region
The diagonal cross peaks are marked as black
signals labeled 1 to 9. The red cross peaks are
sequential NOEs.
If 2Val (V), 5 from a tryptophan (W) and 9 from
a glycine (G), we can write that sequence
is XVXXXWXXG or GXXWXXXVX 983567421 sequence of
signals GAKWSRYVP amino acid sequence
1 ? 2 ? 4 ? 7 ? 6 ? 5 ? 3 ? 8 ? 9
Or reverse
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Sequential Assignment of Ha in residue (i) to NH
in residue (i1) by NOE
Superimposition of a COSY spectrum with blue
annotated cross peaks (through bond) and NOESY
spectrum with red cross peaks. Sequential NOE
between Ha in residue (i) to HN in residue (i1).
CH1/NH5
6 ? 4 ? 1? 5 ? 7 ? 3 ? 2
CH4/NH1
CH6
NH6
NH4
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NOE-distance
using distance constraints to calculate a
structure. In the upper figure is shown a linear
strand with beads. The green, red and blue pairs
of beads, respectively have been shown to be
close to each other. The structure below
represents one solution to determining the
structure based on the three pieces of distance
information.
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Coupling values to setup experiments
140 Hz
H
15 Hz
11 Hz
55 Hz
13C
13C
15N
13C
15N
O
O
H
H
13C
90-100 Hz
30-40 Hz
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Some 3D sequences
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3D-HMQC-COSY
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HMQC-NOESY
NH 1
2
Ha1
Ca1
2
3
Ha2
Ca2
3
4
Ca3
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HMQC-NOESY
R. R. Ernst (nobel lecture 92)
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3D-HMQC-NOESY
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2D-NOESY vs 3D-HMQC-NOESY
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NOESY-HMQC
Ha-1
Ha1
NH1
Ha2
NH2
Ha1
Ha3
Ha2
NH3
L.E.Kay, D.Marion, A.Bax, J.Magn.Reson.,84, 72
(89)
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NOESY-HMQC and HMQC-NOESY
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GE-HMQC-TOCSY
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2D and 3D HMQC-TOCSY
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3D HCCH-TOCSY
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3D-NMR Puzzle approach
HNCA
HN(CO)CA
HNCO
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3D-NMR Puzzle approach
HA(CA)NH
HACACO
HCA(CO)N
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HNCACB CBCA(CO)NH
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Figure 19. Schematic presentation of the combined
spectrum analysis of the three-dimensional HNCACB
and a CBCA(CO)NH spectra. The 15N axis and the
frames in the 15N dimension are coloured blue.
The1H axis and the frames in the 1H dimension are
coloured red. The 13C axis and the frames in the
13C dimension are green. Two planes dN(i),
top-left, and dN(i1), bottom-left, are
highlighted. The cross peaks in HNCACB are and
in CBCA(CO)NH are
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HNCO Sequential assignment in protein
1JNH 90-100 Hz 1JN-13CO 15 Hz 1JN-13Ca 11
Hz
t1 15N
t2 CO
t3 HN
d 18 ms
Bax et all., J.Magn.Reson., 89, 486-514 (90)
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HNCO 2D and 3D
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HNCA Sequential assignment in protein
1JNH 90-100 Hz 1JN-13CO 15 Hz 1JN-13Ca 11
Hz 2JN-13Ca-1 7 Hz
t3
t
t
t
t
1H
t1/2
t1/2
15N
d
d
t2
13Ca
13CO
t2 Ca
t1 15N
t3 HN
d 1 / 2 JN-Ca 33 ms
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HCACO Sequential assignment in protein
1JCH 140 Hz 1J13Ca -13CO 55 Hz
t3
t
t
t
t
1H
t1/2
13Ca
t1/2
D
D
COSY
t2
13CO
15N
t1 Ca
t3 H Ca
t2 13CO
D 1 / 4 JCa-CO 3 ms
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HCA(CO)N Sequential assignment in protein
1JCH 140 Hz 1J13Ca -13CO 55 Hz 1J15N -13CO
15 Hz
t3
t
t
t
t
1H
t1/2
13Ca
t1/2
D
D
COSY
13CO
d
d
t2
15N
t1 Ca
t3 H Ca
t2 15N
D 1 / 4 JCa-CO 3 ms
d 1 / 3 JCO-N 18-20 ms
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HN(CA)CO and HNCOcomplementary experiments
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Example of HNCA and HN(CO)CA
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Example of sequential assignment
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HN(CO)(CA)
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2D HN(CO)(CA)
NH
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4D-NMR
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