1 resonance assignment strategies. 2 amino acid sequence + the assignment problem

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Resonance assignment Resonance assignment strategiesstrategies

2

Amino acid sequence

+

The assignment problem

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Assignment via 1H NMR

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Proton frequenciesProton frequencies

- Assignment based on backbone HN

➡ present in all residues (except Proline) ➡ unique region of spectrum ➡ well-dispersed resonances

-Scalar couplings (COSY / TOCSY) ➡ identify spin systems (i.e. amino acid type)

- number of resonances (i.e. protons)-frequency of resonances

-Connect with NOESY

Spin Systems

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• Dipolar interaction (NOEs)

➡ through-space contacts

➡ intra-residual, sequential (& long-range) contacts

➡ link spin-systems: identify i & i-1

residue "i -1" residue "i"

1H - 1H NOE

intra-residual NOEs

sequential NOEs

residue "i+1"

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Peptide sequenceR1-A2-Q3-L4-A5-M6-S7

ExampleIntra residueInter residue

1

2

3

4

5

6

7

8

9

8

Example: who is who?Intra residueInter residue

???

Peptide sequenceR1-A2-Q3-L4-A5-M6-S7

1

2

3

4

5

6

7

8

9

9

Example: identify protons & frequencies Intra residue

Inter residue

Peptide sequenceR1-A2-Q3-L4-A5-M6-S7

1

2

3

4

5

6

7

8

9

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Example: assign strips to residuesIntra residueInter residue

C = Ala

Peptide sequenceR1-A2-Q3-L4-A5-M6-S7

1

2

3

4

5

6

7

8

9

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Example: assign strips to residuesIntra residueInter residue

C = AlaB = Leu

Peptide sequenceR1-A2-Q3-L4-A5-M6-S7

1

2

3

4

5

6

7

8

9

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Example: assign strips to sequence Intra residue

Inter residue

C = A2 / A5B = L4A = Q3 / M6

PossibilitiesI: A2 - Q3 - L4 (CAB) II: Q3 - L4 - A5 (ABC)III: L4 - A5 - M6 (BCA)

Peptide sequenceR1-A2-Q3-L4-A5-M6-S7

1

2

3

4

5

6

7

8

9

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Example: connect residues

• Use HN-HN NOEs

➡B has cross-peaks to both A & C

➡ABC

➡Q3 - L4 - A5

Peptide sequenceR1-A2-Q3-L4-A5-M6-S7

PossibilitiesI: A2 - Q3 - L4 (CAB) II: Q3 - L4 - A5 (ABC)III: L4 - A5 - M6 (BCA)

1

2

3

4

5

6

7

8

9

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Example: verify!!!

• Use HN-Hα NOEs to verify

➡sequential HN(i) - Hα(i-1)

- HN(C) - Hα(B)

- HN(B) - Hα(A)

➡ABC

➡Q3 - L4 - A5

Peptide sequenceR1-A2-Q3-L4-A5-M6-S7

PossibilitiesI: A2 - Q3 - L4 (CAB) II: Q3 - L4 - A5 (ABC)III: L4 - A5 - M6 (BCA)

1

2

3

4

5

6

7

8

9

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Assignment via Assignment via 11H, H, 1515N, and N, and 1313CC

J coupling constants

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• Heteronuclear experiments

➡more information

➡ increase resolution: 2D → 3D → 4D ...

➡sequential assignment based on scalar coupling

Triple resonance NMR

• Advantages

➡ through-bond (J) magnetization transfer to neighboring residues (instead of NOE)

➡ 1J scalar coupling much larger than 3JHH (<10 Hz) (efficient transfer of magnetization)

Protons

Other nuclei 13C, 15N

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Nomenclature

Names of scalar experiments based on atoms detected

HNCA HN(CO)CAHN(CA)CO HNCO

HN(CA)CB HN(COCA)CB

Pairs of experiments distinguish between intra-residual and sequential resonances

Residuei-1 & i i-1 i-1 & i i-1

i-1 & i i-1

Example: 3D HNCA

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Example: analyze frequencies

7.71

122.8

a

8.40

123.8 61.3

2

58.52

b

--15N–13Cα–13C–15N–13Cα–13C--

H

R

H

R

H H

O O

--15N–13Cα–13C–15N–13Cα–13C--

H

R

H

R

H H

O O

8.24

117.1 55.0

3

68.43

68.43

c

--15N–13Cα–13C–15N–13Cα–13C--

H

R

H

R

H H

O O

61.32

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• Numerically...

➡c: Cα (i) = a: Cα (i-1)

➡a: Cα (i) = b: Cα (i-1)

Example: link the spin-systems

Sequence: c – a – b Sequence: c – a – b

Example: link the spin-systems

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13Cα

1HN

15N

i-1

i & i-1

HNCA versus HN(CO)CA

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Assigned [1H-15N]-HSQC

15N

1H

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• If no label or only 15N: NOESY / TOCSY

• Identify spin-system in TOCSY

• Sequential NOEs to link spin-systems

• 13C & 15N: 3D triple resonance experiments

• Sequential information through bond (J coupling)

• HNCA / HN(CO)CA (and many more)

Key concepts assignment

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NMR observablesNMR observables&&

structural restraintsstructural restraints

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Protein structure• Secondary structure

- alpha helix, beta-sheet, etc.

• Tertiary structure

- full 3D structure

• Experimental data that give information about the protein structure

- NMR observables

• Translate the experimental data into parameters that can be used in a structure calculation

- Structural restraints

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NMR observables vs. structural restraints

- 3J-coupling dihedral angle- Chemical shifts secondary structure- NOE’s H-H distances- Paramagnetic relaxation enhancement (PRE) distances- Residual dipolar coupling (RDC) orientation of vectors- H/D exchange hydrogen bonds

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Karplus relation:

J = A.cos2(φ) + B.cos (φ) + C

measured 3J(HNHα)

reports on φ

φ

OBSERVABLE: homonuclear J-couplings

φ

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φ

ω ~ 180º

N NC C C C

C C

O Oψ ω

RESTRAINTS: dihedral angles

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• 13Cα and 13Cβ chemical shifts

- sensitive to dihedral angles

- report on secondary structure elements

OBSERVABLE: chemical shift

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Chemical Shift Index (CSI)

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Predicting dihedral angels: TALOS

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anti-parallel β-strand α-helix

φ -130 -60

ψ 125 -45

β-strand α-helix

RESTRAINTS: dihedral angles

φ ψ

ψ

φ

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+180

ψ

-180

-180 φ +180

α-helix

β-strand

Ramachandran plot

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• 1H-1H NOEs

- signal intensity proportional to 1/r6

- reports on distance between protons

➡ distance restraints (up to 5-6 Å)

Sequential

A B C D Z• • • •

Intra-residue

(used for identifying spin-

systems) Medium range

Sequential & medium range NOEs - SECONDARY STRUCTURE

OBSERVABLE: NOE

Longe range

r =

r =

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RESTRAINT: distances

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NOEs in secondary structure elements

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Short distances in β-strands

anti-parallel

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NOEs in secondary structure elements

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Short distances in α-helices

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Short distances in α-helices

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OBSERVABLE: PRE

• paramagnetic relaxation enhancement (PRE)

• paramagnetic center (unpaired electron)

- radical (e.g. nitroxide)

- certain metal ions (i.e. Mn2+, Gd3+)

• nuclear spin relaxation is enhanced by the paramagnetic center

- signals will broaden (or even disappear)

- effect is dependent on the distance to the paramagnetic center

➡ 1/r6

- because of the large magnetic moment of the unpaired electron the PRE provides long-range distance information (Mn2+ ~35 Å)

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OBSERVABLE: Residual dipolar couplings

Dipolar coupling

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Residual dipolar coupling (RDC)

Dipolar coupling

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Residual dipolar coupling (RDC)

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RDC reports on orientation of bond-vector - orientation of bond-vector within a molecular

alignment tensor (defined by Aa and Ar) with respect to the magnetic field

Long range orientational restraint - TERTIARY STRUCTURE

RDC: Orientational restraint

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The more RDCs, the better...

N NC C C C

C C

O O

• RDCs commonly measured

- 1D 1HN-15N

- 1D 13C ’-15N

- 1D 13Cα-13C ’

- 1D 1Hα-13Cα

• In perdeuterated proteins

- 2D 1HN-13C ’

- 2D/3D 1HN-13Cα

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RESTRAINT: RDC Orientation

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OBSERVABLE: H/D exchange rates

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• OBSERVABLES

- chemical shifts (1H, 15N, 13C, ...)

- J-couplings, e.g. 3J(HN,Hα)

- medium-range NOEs

- hydrogen/deuterium exchange

Sources of structural information

- long-range NOEs

- residual dipolar couplings (RDCs)

- paramagnetic relaxation enhancement (PREs)

Secondary structure

Tertiary structure