nmr spectroscopy lecture 8 basics and applications in biology

NMR Spectroscopy

Lecture 8

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: http://web.mit.edu/speclab/www/PDF/DCIF-IntroNMRpart1-theory-o07.pdf

For YouTube fans: http://www.youtube.com/watch?v=uUM5BNBULwc

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

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

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

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

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

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

ppm

H2O

Protein NMR

Folded versus unfolded protein

folded

unfolded

Different chemical environments cause different chemical shifts

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

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

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

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

NOESY example

NOESY -Symmetrical about diagonal-Diagonal peaks correspond to 1D NMR spectrum

chentobiose

Protein NMR spectra

NOESY -In principle sufficient information to calculate the 3D structure of the protein

3D NMR spectra

For proteins enriched with 15N and 13C

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

and more…

Paul Lauterbur

Medicine: MR imaging

2003Peter Mansfield

3D structures of proteins by NMR

NOESY spectrum 3D structure

Each NOESY cross-peak presents a distance restraint

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

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 )

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

Mobility

NMR works in solution-Can measure conformational exchange-Different experiments for different time scales

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

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

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

Finally

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