Biomolecular Nuclear Magnetic Resonance Spectroscopy

1. 01/21/04 Biomolecular Nuclear Magnetic Resonance Spectroscopy BASIC CONCEPTS OF NMR • How does NMR work? • Pulse FT NMR • Resonance assignment NMR text: Chapter 22 in Protein and Peptide Drug Analysis “Solution Structure Determination of Proteins by NMR”

2. NMR in Medicine and Biology • MRI- Magnetic Resonance Imaging (water) • In-vivo spectroscopy (metabolites) • Solid-state NMR (large structures) • Solution NMR • Bioanalytical, primary structure • Three-dimensional structure • Molecular motions • Molecular interactions- binding, reactions Ligand screening (Pharma)

3. Nuclear Spin • Nuclear spin angular momentum is a quantized property of the nucleus in each atom, which arises from the sub-atomic properties of neutrons and protons • The nuclear spin angular momentum of each atom is represented by a nuclear spin quantum number (I) • All nuclei with odd mass numbers have I=1/2,3/2... • Nuclei with even mass numbers and an even number of protons have I=0 • Nuclei with even mass numbers and an odd number of protons have I=1,2,3… Biomolecular NMR: primarily spin 1/2 nuclei (1H, 13C, 15N, 31P)

4. Efficiency factor- nucleus DE = h g Ho Constants Strength of magnet Nuclei With Non-Zero SpinAlign in Magnetic Fields Ho Alignment Energy parallel anti-parallel

5. + - + - + - + - + - + - + - + - + - + - + - + - NMR: The Bar Magnet Analogy p p p ap ap ap + - + - + - Ho = + - 1. force non-alignment 2. release

6. p 1. equilibrium DE Efficiency factor- nucleus ap H1 DE = h g Ho hn = DE 2. pump in energy Constants Strength of magnet p 3. non-equilibrium ap Resonance: Perturb Equilibrium Ho

7. p DE 3. Non-equilibrium ap hn = DE 4. release energy (detect) p 5. equilibrium ap Return to Equilibrium (Relax): Read Out Signals

8. Sensitivity (S) ~ D(population) Efficiency factor- nucleus Np Nap -DE/kT = e S ~ DN = DE = h g Ho Constants Strength of magnet Magnetic Resonance Sensitivity DE is small At room temp., DN ~ 1:105 Intrinsically low sensitivity Need lots of sample Increase sensitivity by increasing magnetic field strength

9. Intrinsic Sensitivity of Nuclei Nucleusg % Natural Relative Abundance Sensitivity 1H 2.7 x 108 99.98 1.0 13C 6.7 x 107 1.11 0.004 15N -2.7 x 107 0.36 0.0004 31P 1.1 x 108 100. 0.5

10. Two spins All spins  Sum Bulk Magnetization excess facing down The Classical Treatment:Nuclear Spin Angular Momentum Ho parallel anti-parallel • Torque + int. motion = precession • Precession around Z axis • Larmor frequency (): DE = hgHo  DE = hn  n = H0 = 

11. Pulse Fourier Transform NMR t 90ºx RF pulse =  =  H0 Ho Ho A t Fourier Transform f  Variation of signal at X axis vs. time NMR frequency

12. The Power of Fourier Transform t 90ºx RF pulse + 1 =  H0 2 =  H0 A t Fourier Transform f 2 1 • NMR frequency domain • Spectrum of frequencies • NMR time domain • Variation in amplitude vs time

13. 90º pulse Experiment (t) equilibration detection of signals Fourier Transform Data Analysis Time domain (t) The Pulse FT NMR Experiment

14. NMR TerminologyChemical Shift & Linewidth The exact resonance frequency (chemical shift) is determined by the electronic environment of the nucleus

15. NMR Scalar and Dipolar Coupling Through Space Through Bonds • Coupling of nuclei gives information on structure

16. OH CH2 CH3 The key attribute: use the scalar and dipolar couplings to match the set of signals with the molecular structure Resonance Assignment CH3-CH2-OH Which signal from which H atoms?

17. Proteins Have Too Many Signals! 1H NMR Spectrum of Ubiquitin ~500 resonances • Resolve resonances by multi-dimensional experiments