3.6.2008 Lecture 6 • heteronuclear assignment • Scripte: homepage Anne Ulrich-Gruppe ÆLehre ÆEinführung in die biomolekulare NMR Æ 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)
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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
3.6.2008
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