nmr – a versatile tool in structural proteomics · a 2d nmr noesy spectrum of a protein depicts...
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NMR – a versatile tool in structural proteomics
NMR basics Protein spectra, multidimensional NMR
Resonance assignments Structure determination
Ligand mappning Folding
Dynamics
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NMR fundamentals from a quantum mechanical perspective
Certain atomic nuclei possess a property called ’spin’, characterized by the spin quantum number I.
If we put these nuclei in a magnetic field, they will adopt different states with different energies.
I= ½ gives two states: ψ = α , ψ = β
1H: I=½ 12C: I=0 14N: I=1 16O:I=0 31P: I= ½ 2H: I=1 13C: I= ½ 15N:I= ½ 17O: I=5/2
ε
Increasing magnetic field Bo
Δε = hν Energy difference is small => radio freq
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Equipment for NMR
Supraconducting, nitrogein/heliumfilled magnets
Pulse- and wave form generators, gradient unit, general electronics
User terminal, pulse programming unit
Probes
Samples
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A spectroskopic method where we can study atoms in various amino acids!
… but how do we know which signals belong to which atom???
SÄTT IN SPEKTRUM
Backbone NH
Aromatic Hs
Hα
Aliphatic Hs
Methyl Hs
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Resonance assignment requires evaluation of multidimensional spectra
Folding
Ligand bindning
Structure Dynamics
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Nuclear spins affect each other - using 2D experiments we can analyse how this is done!
Magnetisation transfer through space – distance dependent (< 5Å), ~ 1/r6
coherence Spinn relaxation (nOe)
Magnetisation transfer between nuclear neighbors
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Magnetic labelling: requires gene technology and bacterial
overexpression 1H: I=½ 12C: I=0 14N: I=1 16O:I=0 31P: I= ½ 2H: I=1 13C: I= ½ 15N:I= ½ 17O: I=5/2
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A 2D spectrum of the aromatic part of an organic compound
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A 2D NMR NOESY spectrum of a protein depicts intermolecular distances between all protons in the biomolecule.
A protein!
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3D-NOESY-HSQC
HSQC-sidan
1HN
1H
15N
1HN
15N
The HSQC experiment can be recorded and analyzed separately.
HSQCs show one peak per amino acid.
HSQC
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15N Dispersed 1H-1H TOCSY
3 overlapped NH resonances
Same NH, different 15N
F1
F2 F3
1H 1H 15N
t1 t2 t3
TOCSY HSQC
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Basic strategy for NMR assignment of proteins:
1. Identify NMR-resonances for each amino acid:
2. Put them in order: Sequence-specific assignment
1 2 3 4 5 6 7 R - G - S - T - L - G - S
L T G S S R G
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Heteronuclear assignment:
Exempel: HNCA!
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Other backbone experiments
Names of experiments relate to magnetization transfer pathways
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Multiple redundancies gives plenty of back-check options !
Sidechain assignments are required for structure determination by NOEs
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Kurt Wüthrich
Nobel laureate 2002 ETH, Zürich
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Secondary structure elements can be identified by unique short distances α-helix
(CαH)i – NHi+3, (i+2, i+4)
(CαH)i - (CβH)i+3
β-sheet
NH – NH across the sheet
CαH – CαH across the sheet
CαHi – NHi+1 along the chain
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Ηα-NH
Ηα,NHi+3
Ηα,NHi+2
Ηα,NHi+1 Hα
5 Å
NH
Hα R1
O
NH
Hα R2
O
NH
Hα R3
O
NH
Hα R4
O
NH-Hα
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Ηα-NH
Ηα,NHi+3
Ηα,NHi+2
Ηα,NHi+1 Hα
5 Å
NH
Hα R1
O
NH
Hα R2
O
NH
Hα R3
O
NH
Hα R4
O
NH-Hα
Long-range NOEs!
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This is the location of the helix in the Grx4- structure!
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NMR-structures a family of structures consistent with the NMR-derived experimental constraints set (distances, angles, chemical shifts etc)
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First generation structures: ~7 NOEs per residue rmsd 1.5 bb / 2.0 all atoms Second generation structures: ~10 NOEs per residue rmsd 0.9 bb / 1.2 all atoms Third generation structures: ~13 NOEs per residue rmsd 0.7 bb / 0.9 all atoms Fourth generation structures: ~16 or more NOEs per residue rmsd 0.4 bb / 0.9 all atoms rmsd 0.5 for ordered side chains
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WR41
C-TmZip
ER14
MMP-1
IL13
FGF-2
WR90
WR64
LC8
ER115
JR19
ZR18
OP3
WR33
ZR31 Z-domain
IR24
Examples of NESG structures
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Problem vid högre molekylvikter
• Långsam rörelse i lösning gör att signalen relaxerar snabbare → dåligt signal-brus och breda signaler
• Växelverkan mellan relaxerande kärnor ökar relaxationen • Överlappande toppar även i 2D och 3D spektra
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Var går gränsen för NMR på proteiner?
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Malate synthase G (MSG) from E. coli - a monomeric 723-residue protein 1531 NOE, 1101 dihedral angles, 415 residual dipolar coupling, and 300
carbonyl shift restraints. Tugarinov, Choy, Orekov and Kay, PNAS 2005
Är subdomänerna rörliga? Kan vi utvärdera det med NMR?
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Folded or unfolded protein?
1D-spektra HSQC
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Ligand bindning studied in HSQC spectra
Binding of the tumor inhibitor chalcone to the p53 interacting protein MDM2; spectra with different ligand concentration are colored differently
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15N-relaxation gives information on dynamics in proteins – active sites are flexible!
DHFR, Dyson,Wright and coworkers
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NMR - treats J • High-resolution structures can be determined
also for non-crystallizable proteins • Rapid and specific mapping of ligand binding
and protein interactions • Dynamics studies But: • Technically advanced • Specific sample requirements • Current convenient max size limit around 35
kDa