NMR Lecture6
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Transcript of NMR Lecture6
3.6.2008
Lecture 6
• heteronuclear assignment
• Scripte: homepage Anne Ulrich-Gruppe Lehre Einführung in die biomolekulare NMR
lecture 1 und 2 • http://www.cis.rit.edu/htbooks/nmr/ The basics of
NMR – HTML book by J.P. Hornak • James Keeler NMR lectures (googlen)
3.6.2008
Heteronuclear backbone assignment
1H - spectroscopy and 15N-spectroscopy• Rely on TOCSY-NOESY comparison to
join adjacent amino acids, uses 2J and 3J-couplings
13C/15N heteronuclear assignment• Combines adjacent amino acids through J-
coupling between carbons and nitrogen
3.6.2008
Spin system assignment based on J-correlations between heteroatoms
1J and 2J-couplings
to transfer magnetization
• from spin 1• to spin 2 • to spin 3• to …..
Sattler et al. Prog. NMR Spectrosc. (1999) 34, 93-158.
Main experiments for backbone assignment: HNCA/HN(CO)CA -- HNCACB/HN(CO)CACB
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Backbone assignment
O
C Cα
Cβ
N
HN
C
O
N
HN
1JNCα
2JNCα
1JHNN
7 Hz12 Hz
95 Hz
1JHNN95 Hz O
C Cα
Cβ
N
HN
C
O
N
HN
1JCαC’ 1JNC’
40 Hz13 Hz
1JHNN95 Hz
HN(CO)CAHNCA
Way of magnetization can be controlled through selective pulses specific for the respective nuclei. Independent of structure (at a first approximation) Transfer efficiencies depend on size of the J-couplings
3.6.2008
HNCA & HN(CO)CA
Cα(i-1), N(i), HN(i)
HN(CO)CA:
HNCA:
13C
1H
15N
13C
ω1H,ω15N,ω13C
1H,15N
Cαi-1
Cαi
Cα(i-1), Cα(i) N(i), HN(i)
3.6.2008
Additional information
3.6.2008
HNCACB & HN(CO)CACB
1H
15N
13C
ω1H,ω15N,ω13C
1H,15N
Cαi-1
Cαi
Cβi-1
Cβi
Cβ(i-1), Cα(i-1), N(i), HN(i)
HN(CO)CACB:
HNCA:
Cβ(i-1), Cα(i-1), Cβ(i),Cα(i), N(i), HN(i)
3.6.2008
HNCACB - Strips
1H
15N
1H,15N
15N
1H
ω15N1,ω1H
1H
13C
ω1H,ω15N,ω13CCαi-1
Cαi
Cβi-1
Cβi
3.6.2008
Extract Strips at HN,N frequency
CαI-1 Cβ−1i
Cαi Cβi
EAYVIE
15N
1H
ω1H1,ω15N1
(E)A1H,15N
(Y)V1H,15N
(V)I1H,15N
(A)Y1H,15N
1H,15N HSQC
Carbon chemical shifts are characteristic for residue
type
(I)E1H,15N
13C
70 ppm
20
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C’ CA CB CG CDALA 178.5 52.82 19.26ASN 176.1 53.33 39.09 177.3ASP 175.9 52.99 38.33 177.4ARG 177.1 56.48 30.93 27.33 43.55CYS 175.3 58.63 28.34GLN 176.8 56.22 29.53 33.96 180.5GLU 176.8 56.09 28.88 32.88 180GLY 174.9 45.39HIS 175.1 55.39 29.12 131.4 120.2ILE 177.1 61.62 38.91 27.46, 17.47 13.16LEU 178.2 55.47 42.46 27.11 24.99, 23.32LYS 177.4 56.71 33.21 25.01 29.33MET 177.1 55.77 32.94 32.25 PHE 176.6 58.09 39.75 139.2 132PRO 177.8 63.7 32.22 27.32 49.81SER 175.4 58.67 64.06THR 175.6 62.01 70.01 21.6TRP 177.1 57.6 29.75 111.7 127.4, 129.6 TYR 176.7 58.28 38.94 130.8 133.3VAL 177 62.61 32.82 21.11, 20.34
13C chemical shifts
3.6.2008
Match Strips at Cα, Cβ frequency
(E)A1H,15N
CAi
CBi
CAi-1
CBi-1
EAYVIE
(A)Y1H,15N
(Y)V1H,15N
(V)I1H,15N
15N
1H
ω1H1,ω15N1
1H,15N HSQC
(I)E1H,15N
13C
70 ppm
20
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LIM1 (MLP)
• gly arg ser phe his lys thr cys phe his cysmet ala cys arg lys ala leu asp
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Overlay HNCACB with CACB(CO)NH
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Overlay HNCACB with CACB(CO)NH and HNCA
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Side chain assignment
3.6.2008
CCONH
(E)A1H,15N
CAi-1
CBi-1
EAYVIE
(A)Y1H,15N
(Y)V1H,15N
(V)I1H,15N
15N
1H
ω1H1,ω15N1
1H,15N HSQC
CGi-1
CDi-1
(I)E1H,15N
13C
70 ppm
20
5
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Overlay CCONH with CACB(CO)NH
3.6.2008