Title: Sequence Assignments
1Sequence Assignments
Secondary Structure
Tertiary Structure
2Sequence Assignments
COSY NOESY COSY-NOESY walk Example Spin Systems
Basic techniques
3D-NMR Experiments HSQC TOCSY HSQC-TOCSY Arranging
3D Slices
Advanced techniques
3Structure 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
4COSY - 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
5Structure 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
6NOESY - 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
7Structure 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
8Structure 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
9Amino Acid Spin Systems
10Amino Acid Spin Systems
H
O
Alanine
C
N
C
H
H
bCH3
aH
11Amino Acid Spin Systems
H
O
Glycine
C
N
C
H
H
aH
aH
12Amino Acid Spin Systems
OH
Ser, Cys, Asp, Asn Phe, Tyr, His, Trp
O
C
N
C
H
H
bH
bH
aH
13Amino Acid Spin Systems
H
CH3 - C - CH3
O
Leucine
C
N
C
H
H
dCH3
dCH3
gH
bH
bH
aH
14Three-dimensional NMR
15General 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
16General Three Dimensional Experiment
13C or 15N chemical shift
1H chemical shift
1H chemical shift
2D planes or slices in 3D spectrum.
17TOCSY - 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
18HSQC - 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
19Sequence Assignments
Secondary Structure
Tertiary Structure
20Secondary Structure
Secondary structure from Chemical
shifts Dipolar couplings J-couplings and
torsion angles Measuring NOEs Measuring
J-couplings
21Chemical shifts (1H, 13C, 15N) are sensitive to f
and y torsion angles
Cb
H
Ca
N
C
H
H
O
H
N
22Chemical 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
23Secondary 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
24Secondary 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
25Secondary 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
26Secondary 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
27Secondary Structure
Sequence MALRRVETTYGDAWCSTQNLIVWRSTERLN dN
N daN daN(i,i3) 3JHNa
helix
sheet
sheet
3JHNa gt 7 Hz
28Measuring 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