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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” 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) 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) Nuclei With Non-Zero Spin Align in Magnetic Fields Efficiency factornucleus Ho Alignment parallel anti-parallel Energy DE = h g Ho Constants Strength of magnet NMR: The Bar Magnet Analogy p p ap ap ap + - + - + - 1. force non-alignment + - + - + - - + - = + - + - Ho + - + + - 2. release + - + - + - + - p Resonance: Perturb Equilibrium p Ho DE 1. equilibrium Efficiency factornucleus ap H1 hn = DE DE = h g Ho 2. pump in energy Constants p ap 3. non-equilibrium Strength of magnet Return to Equilibrium (Relax): Read Out Signals p DE 3. Non-equilibrium ap hn = DE 4. release energy (detect) p 5. equilibrium ap Magnetic Resonance Sensitivity Sensitivity (S) ~ D(population) S ~ DN = Np = e-DE/kT Nap DE is small At room temp., DN ~ 1:105 Intrinsically low sensitivity Need lots of sample Efficiency factornucleus DE = h g Ho Constants Strength of magnet Increase sensitivity by increasing magnetic field strength Intrinsic Sensitivity of Nuclei Nucleus g % Natural Abundance Relative Sensitivity 1H 2.7 x 108 99.98 13C 6.7 x 107 1.11 0.004 15N -2.7 x 107 0.36 0.0004 31P 1.1 x 108 100. 1.0 0.5 The Classical Treatment: Nuclear Spin Angular Momentum Two spins All spins Sum Ho parallel anti-parallel Torque + int. motion = precession Precession around Z axis Larmor frequency (): DE = hgHo DE = hn n = gH0 = excess facing down Bulk Magnetization Pulse Fourier Transform NMR t 90ºx RF pulse = = g H0 Ho Ho A t f NMR frequency Fourier Transform Variation of signal at X axis vs. time The Power of Fourier Transform t 90ºx RF pulse + 1 = g H0 2 = g H0 A t f 2 1 Fourier Transform NMR frequency domain NMR time domain Spectrum of frequencies Variation in amplitude vs time The Pulse FT NMR Experiment 90º pulse Experiment (t) equilibration detection of signals Fourier Transform Data Analysis Time domain (t) NMR Terminology Chemical Shift & Linewidth The exact resonance frequency (chemical shift) is determined by the electronic environment of the nucleus NMR Scalar and Dipolar Coupling Through Space Through Bonds Coupling of nuclei gives information on structure Resonance Assignment CH3-CH2-OH OH CH2 CH3 Which signal from which H atoms? The key attribute: use the scalar and dipolar couplings to match the set of signals with the molecular structure Proteins Have Too Many Signals! 1H NMR Spectrum of Ubiquitin ~500 resonances Resolve resonances by multi-dimensional experiments 2D NMR: Coupling is the Key 2D detect signals twice (before/after coupling) 90º pulse t1 Same as 1D experiment Transfers between coupled spins 2D NMR Pulse Sequence t2 t1 t2 2D NMR Spectrum Pulse Sequence t1 Spectrum Before mixing After mixing Coupled spins t2 The Power of 2D NMR: Resolving Overlapping Signals 1D 2 signals overlapped 2D 2 cross peaks resolved Acronyms For Basic Experiments Differ Only By The Nature Of Mixing Scalar Coupling Dipolar Coupling Homonuclear Heteronuclear COSY HSQC TOCSY Hetero-TOCSY Multiple Quantum HMQC NOESY NOESY-HSQC NOESY-HMQC Multi-Dimensional NMR: Built on the 2D Principle 3D- detect signals 3 times 90º pulse (t3) t1 Same as 1D experiment t2 t3 3D NMR Pulse Sequence Experiments are composites acronyms are composites