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Lecture 8 NMR Spectroscopy Basics and applications in biology.

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1 Lecture 8 NMR Spectroscopy 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
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 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 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:

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:

9 Metabolomics 13C-1H correlation - Greatly improved spectral resolution
H2O 13C 1H From:

10 Protein NMR H2O ppm

11 Folded versus unfolded protein
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 15N 1H

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

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% 500 MHz 950 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,

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 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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