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Biomolecular Nuclear Magnetic Resonance Spectroscopy

01/15/03. Biomolecular Nuclear Magnetic Resonance Spectroscopy. BASIC CONCEPTS OF NMR How does NMR work? Pulse FT NMR 2D NMR experiments nD NMR experiments. NMR text: Chapter 22 in Protein and Peptide Drug Analysis “Solution Structure Determination of Proteins by NMR”.

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Biomolecular Nuclear Magnetic Resonance Spectroscopy

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  1. 01/15/03 Biomolecular Nuclear Magnetic Resonance Spectroscopy BASIC CONCEPTS OF NMR • How does NMR work? • Pulse FT NMR • 2D NMR experiments • nD NMR experiments 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 Ho Alignment Energy parallel DE = h g Ho Constants Strength of magnet anti-parallel Nuclei With Non-Zero SpinAlign in Magnetic Fields

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

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

  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 Ho parallel anti-parallel Bulk Magnetization excess facing down The Classical Treatment:Nuclear Spin Angular Momentum • 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

  18. t1 t2 t1 t2 2D NMR: Coupling is the Key 2D detect signals twice (before/after coupling) 90º pulse Transfers between coupled spins Same as 1D experiment 2D NMR Pulse Sequence

  19. 2D NMR Spectrum Pulse Sequence t1 t2 Spectrum Before mixing Coupled spins After mixing

  20. The Power of 2D NMR:Resolving Overlapping Signals 1D 2 signals overlapped 2D 2 cross peaks resolved

  21. Acronyms For Basic ExperimentsDiffer Only By The Nature Of Mixing Homonuclear Heteronuclear Scalar Coupling COSY HSQC TOCSY Hetero-TOCSY HMQC Multiple Quantum Dipolar Coupling NOESY NOESY-HSQC NOESY-HMQC

  22. t2 t1 t3 Multi-Dimensional NMR:Built on the 2D Principle 3D- detect signals 3 times 90º pulse (t3) Same as 1D experiment 3D NMR Pulse Sequence • Experiments are composites  acronyms are composites

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