1 resonance assignment strategies. 2 amino acid sequence + the assignment problem
TRANSCRIPT
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Resonance assignment Resonance assignment strategiesstrategies
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Amino acid sequence
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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
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9
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Example: who is who?Intra residueInter residue
???
Peptide sequenceR1-A2-Q3-L4-A5-M6-S7
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2
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4
5
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9
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Example: identify protons & frequencies Intra residue
Inter residue
Peptide sequenceR1-A2-Q3-L4-A5-M6-S7
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Example: assign strips to residuesIntra residueInter residue
C = Ala
Peptide sequenceR1-A2-Q3-L4-A5-M6-S7
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Example: assign strips to residuesIntra residueInter residue
C = AlaB = Leu
Peptide sequenceR1-A2-Q3-L4-A5-M6-S7
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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
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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)
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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)
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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