1. Biomolecular Nuclear Magnetic Resonance Spectroscopy BASIC CONCEPTS OF NMR How does NMR work? Pulse FT NMR 2D NMR experiments nD NMR experiments 01/15/03 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 ( 1 H, 13 C, 15 N, 31 P)

4. Nuclei With Non-Zero Spin Align in Magnetic Fields HoHo anti-parallel parallel Alignment Energy  E = h  H o Efficiency factor- nucleus ConstantsStrength of magnet

5. NMR: The Bar Magnet Analogy +-+- H o = +-+- +-+- +-+- +-+- +-+- +-+- +-+- +-+- +-+- +-+- +-+- +-+- 1. force non-alignment2. release p ap p p +-+- +-+- +-+-

6. Resonance: Perturb Equilibrium HoHo h  E H1H1 2. pump in energy  E = h  H o Efficiency factor- nucleus ConstantsStrength of magnet p ap 1. equilibrium EE p ap 3. non-equilibrium

7. Return to Equilibrium (Relax): Read Out Signals p ap 5. equilibrium h  E 4. release energy (detect) p ap EE 3. Non-equilibrium

8. Magnetic Resonance Sensitivity  E is small At room temp.,  N ~ 1:10 5 Intrinsically low sensitivity  Need lots of sample  E = h  H o Efficiency factor- nucleus ConstantsStrength of magnet N p N ap = e -  E/kT Sensitivity (S) ~  population) S ~  N = Increase sensitivity by increasing magnetic field strength

9. Intrinsic Sensitivity of Nuclei Nucleus  Natural Relative Abundance Sensitivity 1 H2.7 x 10 8 99.98 1.0 13 C6.7 x 10 7 1.11 0.004 15 N -2.7 x 10 7 0.36 0.0004 31 P1.1 x 10 8 100. 0.5

10. The Classical Treatment: Nuclear Spin Angular Momentum HoHo anti-parallel parallel  Torque + int. motion = precession  Precession around Z axis  Larmor frequency (  ):  E = h  H o   E = h  =  H 0 =  Two spins All spins  Sum Bulk Magnetization excess facing down

11. Pulse Fourier Transform NMR HoHo HoHo 90º x RF pulse t   =  H 0 f Fourier Transform NMR frequency Variation of signal at X axis vs. time t A =

12. The Power of Fourier Transform 90º x RF pulse t 11  1 =  H 0  2 =  H 0 f Fourier Transform + 22 NMR frequency domain  Spectrum of frequencies NMR time domain  Variation in amplitude vs time t A

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

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

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

16. Resonance Assignment CH 3 -CH 2 -OH Which signal from which H atoms? OHCH 2 CH 3 The key attribute: use the scalar and dipolar couplings to match the set of signals with the molecular structure

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

18. 2D NMR: Coupling is the Key 2D detect signals twice (before/after coupling) Same as 1D experiment 90º pulse t1t1 t2t2 t1t1 t2t2 Transfers between coupled spins 2D NMR Pulse Sequence

19. 2D NMR Spectrum Pulse Sequence Spectrum Coupled spins Before mixing After mixing t2t2 t1t1

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

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

22. Multi-Dimensional NMR: Built on the 2D Principle 3D- detect signals 3 times Same as 1D experiment 90º pulse t2t2 t1t1 t3t3 3D NMR Pulse Sequence (t3)(t3)  Experiments are composites  acronyms are composites