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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)

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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

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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

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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)

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Additional information

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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)

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HNCACB - Strips

1H

15N

1H,15N

15N

1H

ω15N1,ω1H

1H

13C

ω1H,ω15N,ω13CCαi-1

Cαi

Cβi-1

Cβi

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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

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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

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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

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