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NMR Spectroscopy

Lecture 8. NMR Spectroscopy. Basics and applications in biology. Lecture overview. Basic principles of NMR spectroscopy NMR of small molecules NMR of proteins. NMR needs high magnetic fields. A good introduction into the basic principles of NMR:

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NMR Spectroscopy

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  1. Lecture 8 NMR Spectroscopy Basics and applications in biology

  2. Lecture overview Basic principles of NMR spectroscopy NMRof small molecules NMR of proteins

  3. 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

  4. 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 • 2pn = -gB0 • n: frequency, • g: a constant, • B0: external magnetic field • E: energy A spectrum always shows peaks as a function of frequency

  5. More than a single spin • Chemical shifts • Measured in ppm (“parts per million”) relative to a reference • Different chemical environments cause different chemical shifts 3.6 ppm D 1.2 ppm

  6. 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 • Doublet: 1 coupling partner • Triplet: 2 coupling partners • Quartet: 3 coupling partners D

  7. 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

  8. 2D NMR Two frequency axes (ppm) • Often symmetrical about the diagonal • Correlates peaks in 1D NMR spectra (plotted on the sides) 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 From: http://www.chem.queensu.ca/facilities/nmr/nmr/webcourse/cosy.htm

  9. Metabolomics 13C-1H correlation - Greatly improved spectral resolution H2O 13C 1H From: http://genomics.uni-regensburg.de/site/gronwald-group/research/metabolomics-by-multidimensional-nmr

  10. Protein NMR H2O ppm

  11. Folded versus unfolded protein folded unfolded Different chemical environments cause different chemical shifts

  12. 2D NMR of proteins - HSQC 15N-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 1H 15N

  13. 2D NMR of proteins - HSQC 15N-HSQC spectrum • One peak per backbone amide • Two peaks per side-chain amide 1H O H 15N C Cα R O 1H C 15N 1H

  14. 2D NMR of proteins - HSQC • HSQC = ‘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

  15. 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

  16. NOESY example NOESY • Symmetrical about diagonal • Diagonal peaks correspond to 1D NMR spectrum chentobiose

  17. Protein NMR spectra NOESY • In principle sufficient information to calculate the 3D structure of the protein

  18. 3D NMR spectra For proteins enriched with 15N and 13C

  19. A bit of history Nobel prizes for NMR spectroscopy 1952 1991 2002 Felix Bloch Edward Purcell Richard Ernst Kurt Wüthrich Chemistry: 3D protein structures by NMR Physics: discovery of NMR Chemistry: FT-NMR, 2D NMR

  20. and more… 2003 Peter Mansfield Paul Lauterbur Medicine: MR imaging

  21. 3D structures of proteins by NMR Each NOESY cross-peak presents a distance restraint NOESY spectrum 3D structure

  22. 3D structures are defined by dihedral angles Amide bonds are planar The backbone conformation of each amino acid residue is defined by a f and a y angle Bond lengths and bond angles are known -> 2 degrees of freedom per amino acid backbone

  23. Scalar couplings reflect dihedral angles Karplus curve • 3-bond couplings (1H-C-C-1H) depend on the dihedral angle a • Can be measured also for 1H-N-C-1H (backbone dihedral angle f)

  24. 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

  25. Mobility NMR works in solution • Can measure conformational exchange • Different experiments for different time scales

  26. 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 2 are highlighted in different colours Science 1996, 274, 1531-1534

  27. 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

  28. 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

  29. Finally • If you think that this course has been good for anything at all, PLEASE give us feedback in SELT! • Our vice chancellor evaluates course quality by SELT feedback • No feedback can threaten existence of course…

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