Sequence Assignments

1
Sequence Assignments
Secondary Structure
Tertiary Structure
2
Sequence Assignments
COSY NOESY COSY-NOESY walk Example Spin Systems
Basic techniques
3D-NMR Experiments HSQC TOCSY HSQC-TOCSY Arranging
3D Slices
Advanced techniques
3
Structure Determination by NMR
O
O
O
N
N
C
N
C
N
C
C
C
C
H
H
H
H
H
H
H
Residue 1
Residue 3
Residue 2
4
COSY - Correlated Spectroscopy
1 dimensional spectrum is along the diagonal
Cross peaks are from through-bond correlations
Cross peaks are useful for assigning individual
amino acid spin systems
5
Structure Determination by NMR
NOESY Crosspeaks lt 5 Å Through Space
R
H
R
R
H
H
C
C
C
O
O
O
N
N
C
N
C
N
C
C
C
C
H
H
H
H
H
H
H
6
NOESY - Nuclear Overhauser Enhancement
Spectroscopy
Cross peaks are from through-space correlations
Cross peaks in COSY spectrum appear in NOESY
spectrum
Both NOESY and COSY spectra are symmetric about
the diagonal
Intensity of cross peaks is related to
internuclear distance
7
Structure Determination by NMR
R
H
R
R
H
H
C
C
C
O
O
O
N
N
C
N
C
N
C
C
C
C
H
H
H
H
H
H
H
NOESY

H
1
Chemical
Shift
COSY
H Chemical Shift
1
8
Structure Determination by NMR
R
H
R
R
H
H
C
C
C
O
O
O
N
N
C
N
C
N
C
C
C
C
H
H
H
H
H
H
H
NOESY

H
1
Chemical
Shift
COSY
H Chemical Shift
1
9
Amino Acid Spin Systems
10
Amino Acid Spin Systems
H
O
Alanine
C
N
C
H
H
bCH3
aH
11
Amino Acid Spin Systems
H
O
Glycine
C
N
C
H
H
aH
aH
12
Amino Acid Spin Systems
OH
Ser, Cys, Asp, Asn Phe, Tyr, His, Trp
O
C
N
C
H
H
bH
bH
aH
13
Amino Acid Spin Systems
H
CH3 - C - CH3
O
Leucine
C
N
C
H
H
dCH3
dCH3
gH
bH
bH
aH
14
Three-dimensional NMR
15
General Three Dimensional Experiment
13C or 15N chemical shift
1H chemical shift
1H chemical shift
1H chemical shift
H
H
3D improves resolution of overlapping resonances
O
O
15N
13C
C
15N
13C
C
H
H
H
H
16
General Three Dimensional Experiment
13C or 15N chemical shift
1H chemical shift
1H chemical shift
2D planes or slices in 3D spectrum.
17
TOCSY - Total Correlation Spectroscopy
dCH3
H
gH
CH3 - C - CH3
H
bH
O
O
1H chemical shift
15N
C
15N
C
C
C
H
H
H
H
aH
NH
1H chemical shift
All protons in amino acid spin system are observed
18
HSQC - TOCSY
15N chemical shift 121.2
15N chemical shift 119.3
1
1
1H chemical shift
5
5
8
9
8
9
1H chemical shift
1H chemical shift
19
Sequence Assignments
Secondary Structure
Tertiary Structure
20
Secondary Structure
Secondary structure from Chemical
shifts Dipolar couplings J-couplings and
torsion angles Measuring NOEs Measuring
J-couplings
21
Chemical shifts (1H, 13C, 15N) are sensitive to f
and y torsion angles
Cb
H
Ca
N
C
H
H
O
H
N
22
Chemical shifts (1H, 13C, 15N) are sensitive to f
and y torsion angles
CH3
Ca
N
C
H
H
O
Helix Sheet CO 175 ppm 172 ppm Ca 52
ppm 48 ppm Cb 15 ppm 20 ppm
For alanine
23
Secondary structure from short 1H-1H distances
Cb
H
O
dbN
Cb
H
N
C
Ca
N
C
Ca
N
H
H
H
H
H
O
dNN
daN
24
Secondary structure from short 1H-1H distances
Cb
H
O
dbN
Cb
H
N
C
Ca
N
C
Ca
N
H
H
H
H
H
O
dNN Intense NOEs for helix lt 3.6
Å for a-helix
daN Intense NOEs for b-strand
lt 2.7 Å for b-strand
25
Secondary structure from J-couplings
3JHNa
Cb
H
Ca
N
C
H
H
O
3JHNa 6.4 cos2q - 1.4 cosq 1.9 with q f
- 60
b
10
8
a
3JHNa
6
4
2
-160 -120 -80 -40 0
f
26
Secondary structure from J-couplings
3JHNa
Cb
H
Ca
N
C
H
H
O

• 3JHNa 6.4 cos2q - 1.4 cosq 1.9
• -helix (f -57), 3JHNa 3.9 Hz
• 310helix (f -60), 3JHNa 4.2 Hz
• Antiparallel b sheet, (f -139), 3JHNa 8.9 Hz
• Parallel b sheet, (f -119), 3JHNa 9.7 Hz

27
Secondary Structure
Sequence MALRRVETTYGDAWCSTQNLIVWRSTERLN dN
N daN daN(i,i3) 3JHNa
helix
sheet
sheet
3JHNa gt 7 Hz
28
Measuring NOE Intensities
Cb
H
O
dbN
Cb
H
N
C
Ca
N
C
Ca
N
H
H
H
H
H
O
Cb
H
O
dbN
Cb
H
N
C
Ca
N
C
Ca
N
H
H
H
H
H
O
NOE Intensity Distance Weak 3.0 - 5.0
Å Medium 2.0 - 4.0 Å Strong 2.0 - 2.5 Å