1. Biomolecular Nuclear Magnetic Resonance Spectroscopy BASIC CONCEPTS OF NMR How does NMR work? Resonance assignment Structure determination 01/24/05 NMR text: 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 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)

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

4. The Resonance Experiment HoHo h  E H1H1 Pump in energy (RF transmitter) Equilibrium EE Non-equilibrium Equilibrium h  E Release energy (RF receiver) NMR signals

5. 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

6. 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

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

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

9. NMR Spectrum to 3D Structure Spectrum H H H H H H H HH H H Interactions Structure

10. Challenges For Determining Protein Structures Using NMR Proteins have thousands of signals Assign the specific signal for each atom Thousands of interactions between atoms- also need to be assigned Need to transform from NMR spectrum through interpretation of scalar and dipolar interactions to generate 3D coordinates

11. 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

12. Proteins Have Many Signals 1 H NMR Spectrum of Ubiquitin ~500 resonances  A large number of signals are overlapped

13. A Critical Feature of Protein NMR Spectra Only some nuclei are coupled Each amino acid gives rise to an independent NMR sub-spectrum, which is much simpler than the complete protein spectrum Methods have been developed to extract each sub-spectrum from the whole

14. Basic Strategy to Assign Resonances in a Protein 1.Identify resonances for each amino acid 2.Put amino acids in order - Sequential assignment ( R-G-S,T-L-G-S ) - Sequence-specific assignment 1 2 3 4 5 6 7 R - G - S - T - L - G - S LT G S S R G

15. Critical Features of Protein NMR Spectra The nuclei are not all mutually coupled Regions of the spectrum correspond to different parts of the amino acid Tertiary structure leads to increased dispersion of resonances

16. Regions of the 1 H NMR Spectrum are Further Dispersed by the 3D Fold What would the unfolded protein look like?

17. Proteins Have Overlapped Signals  Resolve resonances by multi-dimensional experiments 1 H NMR Spectrum of Ubiquitin

18. Resolve Peaks By Multi-D NMR A BONUS  regions in 2D spectra provide protein fingerprints If 2D cross peaks overlap  go to 3D or 4D …..

19. Solution to the Protein Challenge 1.Increase dimensionality of spectra to better resolve signals: 1  2  3  4 2.Detect signals from heteronuclei ( 13 C, 15 N) t2t2 t1t1 t3t3

20. Heteronuclear nD NMR 1. Increase dimensionality of spectra to better resolve signals: 1  2  3  4 2. Detect signals from heteronuclei ( 13 C, 15 N) Better resolution of signals/chemical shifts not correlated between nuclei More information to identify signals Lower sensitivity to MW of protein

21. Structure Determination by NMR

22. NMR Experimental Observables Providing Structural Information Backbone conformation from chemical shifts (Chemical Shift Index- CSI): ,  Distance restraints from NOEs Hydrogen bond restraints Backbone and side chain dihedral angle restraints from scalar couplings Orientation restraints from residual dipolar couplings

23. NMR Structure Calculations Objective is to determine all conformations consistent with the experimental data Programs that only do conformational search lead to bad chemistry  use molecular force fields improve molecular properties  Some programs try to do both at once  Need a reasonable starting structure NMR data is not perfect: noise, incomplete data  multiple solutions (conformational ensemble)

24. Variable Resolution of Structures Secondary structures well defined, loops variable Interiors well defined, surfaces more variable Trends the same for backbone and side chains

25. Restraints and Uncertainty  Large # of restraints = low values of RMSD  Large # of restraints for key hydrophobic side chains

26. Assessing the Quality of NMR Structures Number of experimental constraints RMSD of structural ensemble (subjective!) Violation of constraints- number, magnitude Molecular energies Comparison to known structures: PROCHECK Back-calculation of experimental parameters