1 / 29

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:

garren
Download Presentation

NMR Spectroscopy

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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…

More Related