nmr spectroscopy lecture 8 basics and applications in biology
TRANSCRIPT
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NMR Spectroscopy
Lecture 8
Basics and applications in biology
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Lecture overview
Basic principles of NMR spectroscopy
NMR of small molecules
NMR of proteins
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NMR needs high magnetic fields
A good introduction into the basic principles of NMR: http://web.mit.edu/speclab/www/PDF/DCIF-IntroNMRpart1-theory-o07.pdf
For YouTube fans: http://www.youtube.com/watch?v=uUM5BNBULwc
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NMR = nuclear magnetic resonance
1H, 13C and 15N nuclei-have a very small magnetic moment: “spin 1/2”
For a single spin: two energy levels in a magnetic field
2 = -B0
: frequency,: a constant,B0: external magnetic fieldE: energy
A spectrum always shows peaks as a function of frequency
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More than a single spin
Chemical shifts-Measured in ppm (“parts per million”) relative to a reference-Different chemical environments cause different chemical shifts
1.2 ppm
3.6 ppm
D
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More than a single spin
Scalar coupling constants-Measured in Hz (“Hertz”, s-1) -Caused by different spin states of neighboring spins (“parallel”
or “antiparallel” to B0)-Between spins separated by 1, 2 or 3 chemical bonds
D
- Doublet: 1 coupling partner- Triplet: 2 coupling partners- Quartet: 3 coupling partners
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NMR of urine: metabolomics
Lots of compounds detected simultaneously (“multiplexing”)-Peak integrals are directly proportional to abundance
From: Wang Y et al. PNAS 2008;105:6127-6132
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2D NMR
Two frequency axes (ppm)-Often symmetrical about the diagonal-Correlates peaks in 1D NMR spectra (plotted on the sides)
From: http://www.chem.queensu.ca/facilities/nmr/nmr/webcourse/cosy.htm
Diagonal peaks-Same as 1D NMR spectrum
Cross-peaks-Connect different peaks in 1D NMR spectrum-Arise from scalar couplings or other magnetisation transfer mechanisms
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Metabolomics
13C
1H
H2O
From: http://genomics.uni-regensburg.de/site/gronwald-group/research/metabolomics-by-multidimensional-nmr
13C-1H correlation - Greatly improved spectral resolution
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ppm
H2O
Protein NMR
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Folded versus unfolded protein
folded
unfolded
Different chemical environments cause different chemical shifts
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15N
1H
2D NMR of proteins - HSQC15N-HSQC spectrum-Correlates 15N and 1H NMR spectra-Magnetisation transfer by the scalar coupling between amide nitrogen (15N) and amide proton (1H)-Only cross-peaks, no diagonal peaks
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C
O
Cα15N
1H
R
H
2D NMR of proteins - HSQC15N-HSQC spectrum-One peak per backbone amide-Two peaks per side-chain amide
C
O
15N
1H
1H
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2D NMR of proteins - HSQCHSQC = ‘heteronuclear single-quantum coherence’
Higher magnetic field B0 improves resolution and sensitivity
Protein must be enriched with 15N-Grow E. coli on medium with 15NH4-salt as only nitrogen source-Natural abundance of 15N: 0.3%
950 MHz 500 MHz
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Resonance assignment
Resonance assignment = attribution of a peak in the NMR spectrum to the specific nucleus in the molecule it comes from-Needs a combination of NMR techniques-2D NOESY (NOE spectroscopy) is most important
NOESY-cross-peaks arise from nuclear Overhauser effects (NOEs) between 1H spins
NOEs-arise from through-space dipolar interactions-provide a mechanism for magnetisation transfer-NOE intensity proportional to 1/r6 (r = internuclear distance)-observable for spins closer than ~5 Å
A NOESY cross-peak shows that two 1H spins are in close proximity
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NOESY example
NOESY -Symmetrical about diagonal-Diagonal peaks correspond to 1D NMR spectrum
chentobiose
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Protein NMR spectra
NOESY -In principle sufficient information to calculate the 3D structure of the protein
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3D NMR spectra
For proteins enriched with 15N and 13C
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A bit of history
Nobel prizes for NMR spectroscopy
Kurt WüthrichRichard ErnstFelix Bloch Edward Purcell
Physics: discovery of NMRChemistry: FT-NMR, 2D NMR
1952 1991 2002
Chemistry: 3D protein structures by NMR
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and more…
Paul Lauterbur
Medicine: MR imaging
2003Peter Mansfield
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3D structures of proteins by NMR
NOESY spectrum 3D structure
Each NOESY cross-peak presents a distance restraint
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3D structures are defined by dihedral angles
Amide bonds are planar
The backbone conformation of each amino acid residue is defined by a and a angle
Bond lengths and bond angles are known -> 2 degrees of freedom per amino acid backbone
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Scalar couplings reflect dihedral angles
Karplus curve-3-bond couplings (1H-C-C-1H) depend on the dihedral angle α-Can be measured also for 1H-N-C-1H (backbone dihedral angle )
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NMR structures
The NMR structure of a protein is presented as a bundle of conformers-Each conformer presents a good solution to the NMR restraints-First conformer usually is the best structure-Typically a bundle of 20 conformers is deposited in the PDB
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Mobility
NMR works in solution-Can measure conformational exchange-Different experiments for different time scales
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Drug development
NMR is sensitive to changes in chemical environment-Ligand binding changes the chemical shifts-Sensitive also to weak binding-Gold standard for site-specific ligand binding
Large chem. shift changes induced by compounds 1 and 2are highlighted in different colours
Science 1996, 274, 1531-1534
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Summary I
NMR owes its success to-Long life of the excited magnetisation (seconds)-Low energy (400-1000 MHz = radiofrequency)-Only nuclear spins in a magnetic field can absorb such small energy quanta-High abundance of 1H (99.985%)-Sensitivity to the chemical environment
Drawbacks of NMR-Relatively low sensitivity-Expensive magnets-Hard to become an expert
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Summary II
NMR spectroscopy is the most versatile spectroscopy on earth-Multidimensional
Most powerful analytical tool for chemists-Metabolomics
3D structures of proteins
Mobility information
Ligand binding
MRI
NOT radioactive
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Finally
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