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NMR Spectroscopy Lecture 8 Basics and applications in biology.

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1 NMR Spectroscopy Lecture 8 Basics and applications in biology

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

3 NMR needs high magnetic fields A good introduction into the basic principles of NMR: For YouTube fans:

4 NMR = nuclear magnetic resonance 1 H, 13 C and 15 N nuclei -have a very small magnetic moment: “spin 1/2” For a single spin: two energy levels in a magnetic field 2  = -  B 0 : frequency,  : a constant, B 0 : 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 1.2 ppm 3.6 ppm D

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 B 0 ) -Between spins separated by 1, 2 or 3 chemical bonds D -Doublet: 1 coupling partner -Triplet: 2 coupling partners -Quartet: 3 coupling partners

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:

8 2D NMR Two frequency axes (ppm) -Often symmetrical about the diagonal -Correlates peaks in 1D NMR spectra (plotted on the sides) From: 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

9 Metabolomics 13 C 1H1H H2OH2O From: 13 C- 1 H correlation - Greatly improved spectral resolution

10 ppm H2OH2O Protein NMR

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

12 15 N 1H1H 2D NMR of proteins - HSQC 15 N-HSQC spectrum -Correlates 15 N and 1 H NMR spectra -Magnetisation transfer by the scalar coupling between amide nitrogen ( 15 N) and amide proton ( 1 H) -Only cross-peaks, no diagonal peaks

13 C O CC 15 N 1H1H R H 2D NMR of proteins - HSQC 15 N-HSQC spectrum -One peak per backbone amide -Two peaks per side-chain amide C O 15 N 1H1H 1H1H

14 2D NMR of proteins - HSQC HSQC = ‘heteronuclear single-quantum coherence’ Higher magnetic field B 0 improves resolution and sensitivity Protein must be enriched with 15 N -Grow E. coli on medium with 15 NH 4 -salt as only nitrogen source -Natural abundance of 15 N: 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 1 H spins NOEs -arise from through-space dipolar interactions -provide a mechanism for magnetisation transfer -NOE intensity proportional to 1/r 6 (r = internuclear distance) -observable for spins closer than ~5 Å A NOESY cross-peak shows that two 1 H 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 15 N and 13 C

19 A bit of history Nobel prizes for NMR spectroscopy Kurt WüthrichRichard ErnstFelix BlochEdward Purcell Physics: discovery of NMR Chemistry: FT-NMR, 2D NMR Chemistry: 3D protein structures by NMR

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

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

22 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

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

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,

27 Summary I NMR owes its success to -Long life of the excited magnetisation (seconds) -Low energy ( MHz = radiofrequency) -Only nuclear spins in a magnetic field can absorb such small energy quanta -High abundance of 1 H (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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