1. Biomolecular Nuclear Magnetic Resonance Spectroscopy BASIC CONCEPTS OF NMR How does NMR work? Resonance assignment Structural parameters 01/28/08 Reading: Chapter 22 in Protein and Peptide Drug Analysis “Solution Structure Determination of Proteins by NMR”

2. 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 even mass numbers have I=0,1,2… All nuclei with odd mass numbers have I=1/2,3/2... NMR is possible with all nuclei except I=0, but I=1/2 has simplest physics Biomolecular NMR  primarily 1 H, 13 C, 15 N ( 31 P)

3. Spin 1/2 Nuclei in a Magnetic Field BoBo Energy  E = h  B o Efficiency factor- nucleus ConstantsStrength of magnet

4. 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 Prepare samples enriched in these nuclei

5. Variables Affecting Sensitivity -  E is very small   N is small -  N ~ 1:10 5 (at room T) NMR has low sensitivity  requires lots of sample!  E = h  H o Efficiency factor- nucleus ConstantsStrength of magnet NNNN = e -  E/kT Sensitivity (S) ~  pop. (N  vs N  ) S ~  N = Increase sensitivity by increasing magnetic field strength or reducing electronic noise (cryo-probes)

6. The Resonance Experiment BoBo Equilibrium EE h  E B1B1 Pump in energy (RF transmitter) Non-equilibrium Equilibrium h  E Release energy (RF receiver) NMR signals Strength of signal   (population)

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

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

9. 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

10. 2D NMR Spectra Facilitate Identification of Coupling Coupled spins F1F1 F2F2

11. Biomolecules Have Many Signals 1 H NMR Spectrum of Ubiquitin ~75 residues, ~500 1 H resonances  Terminology: signals are overlapped

12. Challenges For Using NMR to Study Biological Macromolecules Hundreds-thousands of signals! Must assign the specific signal for each atom Thousands of couplings between nuclei- these also need to be assigned

13. Critical Features of Protein NMR Spectra Regions of the spectrum correspond to different parts of the amino acid Tertiary structure leads to increased dispersion of resonances

14. Regions of the 1 H NMR Spectrum and Dispersion by the 3D Fold What would the unfolded protein look like?

15. Critical Features of NMR Spectra of Biomolecules Regions of the spectrum correspond to different parts of the amino acid Tertiary structure leads to increased dispersion of resonances Bio-macromolecules are polymers  The nuclei are coupled to some (but not all!) other nuclei

16. Spectra of Biomacromolecules: Overlapped Sub-Spectra *Groups of coupled nuclei* Each residue in the sequence gives rise to an independent NMR sub-spectrum, which is much simpler than the complete spectrum Methods have been developed to extract each sub-spectrum from the whole

17. Strategy to Assign Resonances in a Protein 1.Identify resonances for each residue (scalar) 2.Put amino acids in order (dipolar) 1 2 3 4 5 6 7 R - G - S - T - L - G - S LT G S S R G Same idea for any biopolymer (e.g. DNA, RNA)

18. Even Sub-Spectra are Overlapped!  Resolve resonances by multi-dimensional experiments 1 H NMR Spectrum of Ubiquitin

19. Use 2D NMR to Resolve Overlapping Signals 1D 2D Sub-spectra overlapped Coupled spins Crosspeaks resolved!

20. Multi-Dimensional NMR If 2D cross peaks overlap  go to 3D or 4D ….. HNHN HH HH

21. Another Solution to the Overlap Challenge 1.Increase dimensionality of spectra to better resolve signals: 1D  2D  3D  4D…. 2.Use attached nuclei ( 13 C, 15 N) to distinguish t2t2 t1t1 t3t3 HAHA HzHz

22. Multi-Dimensional Heteronuclear NMR 15 N- 1 H HSQC F1 Chemical Shift ( 15 N) F2 Chemical Shift ( 1 H)

23. Advantages of Heteronuclear nD NMR Uses a second nucleus to resolve overlap of the first: chemical shift of each nucleus is characteristic/sensitive to different factors More information to identify resonances Less sensitive to MW because this strategy uses large 1 and 2-bond scalar couplings