2. Protein Structure: NMR Spectroscopy Microbiology 343 David Wishart david.wishart@ualberta.ca

3. Objectives To learn about the basic principles of NMR spectroscopy To gain an awareness of what an NMR spectrum looks like and why To gain a basic understanding of how NMR can be used to determine protein structures, along with its strengths/weaknesses

4. NMR Spectroscopy Radio Wave Transceiver

5. Principles of NMR* Measures nuclear magnetism or changes in nuclear magnetism in a molecule NMR spectroscopy measures the absorption of light (radio waves) due to changes in nuclear spin orientation NMR only occurs when a sample is in a strong magnetic field Different nuclei absorb at different energies (frequencies)

6. Electromagnetic Spectrum*

7. Different Types of NMR* Electron Spin Resonance (ESR) –1-10 GHz (frequency) used in analyzing free radicals (unpaired electrons) Magnetic Resonance Imaging (MRI) –50-300 MHz (frequency) for diagnostic imaging of soft tissues (water detection) NMR Spectroscopy (MRS) –300-900 MHz (frequency) primarily used for compound ID and characterization

8. NMR in Everyday Life Magnetic Resonance Imaging

9. NMR Spectroscopy

10. Explaining NMR UV/Vis spectroscopy Sample

11. Explaining NMR*

12. Each NMR Sample Contains 10 23 Atoms with Protons Inside

13. Each Proton (and other nucleons) Has a Spin* Spin up Spin down

14. Each Spinning Proton is Like a “Mini-Magnet”* Spin up Spin down N S N S

15. Radio Waves Are Absrobed by Protons (Cause Flipping) h Low Energy High Energy NN SS

16. The Spins Flip Back & Forth With Different Frequencies Free Induction Decay

17. The Bell Analogy* NH CH CH 3

18. Different Bells (Nuclei) Ring At Different Frequencies*

19. What if You Ring All the Bells At Once? FT

20. How Do You Interpret All This Ringing? - FT NMR* FT Free Induction Decay NMR spectrum

21. Fourier Transformation* F(  ) = f(t)e dt  t Converts from units of time to units of frequency

22. Which Elements or Molecules are NMR Active?* Any atom or element with an odd number of neutrons and/or an odd number of protons Any molecule with NMR active atoms 1 H - 1 proton, no neutrons, AW = 1 13 C - 6 protons, 7 neutrons, AW =13 15 N - 7 protons, 8 neutrons, AW = 15 19 F = 9 protons, 10 neutrons, AW = 19

23. The NMR Equation =  B/2  B = magnetic field strength in Tesla (1 Tesla = 10,000 Gauss = 1000 kitchen magnets)  = gyromagnetic ratio (characteristic of each nucleus, each atom in a molecule

24. Bigger Magnets are Better low frequencyhigh frequency Increasing magnetic field strength

25. Different Isotopes Absorb at Different Frequencies* low frequencyhigh frequency 2H2H 15 N 13 C 19 F 1H1H 30 MHz 50 MHz 125 MHz 480 MHz 500 MHz

26. NMR Magnet

27. Magnet Legs Probe Sample Bore Cryogens Magnet Coil NMR Magnet Cross-Section

28. An NMR Probe

29. NMR Sample & Probe Coil*

30. 1 H NMR Spectra Exhibit...* 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 Chemical Shifts (peaks at different frequencies or ppm values) Splitting Patterns (from spin coupling) Different Peak Intensities (# 1 H)

31. Chemical Shifts* Key to the utility of NMR in chemistry Different 1 H in different molecules exhibit different absorption frequencies Arise from the electron cloud effects of nearby atoms or bonds, which act as little magnets to shift absorption up or down Mostly affected by electronegativity of neighbouring atoms or groups

32. Characteristic Chemical Shifts

33. Spin-Spin Coupling* Many 1 H NMR spectra exhibit peak splitting (doublets, triplets, quartets) This splitting arises from adjacent hydrogens (protons) which cause the absorption frequencies of the observed 1 H to jump to different levels These energy jumps are quantized and the number of levels or splittings = n + 1 where “n” is the number of nearby 1 H’s

34. Spin-Spin Coupling* C - Y C - CHC - CH 2 C - CH 3 H |H | H |H | H |H | H |H | singlet doublet triplet quartet XZXZXZXZ J

35. Spin Coupling Intensities* 1 1 2 1 1 3 3 1 1 4 6 4 1 1 5 10 10 5 1 1 2 Pascal’s Triangle 1 3

36. NMR Peak Intensities* C - CHC - CH 2 C - CH 3 Y |Y | Y |Y | Y |Y | XZXZXZ AUC = 1AUC = 2AUC = 3

37. 1 H NMR Spectrum of a Small Molecule Absorbance

38. 1 H NMR Spectrum of a Large Molecule (Protein)

39. NMR of Big Molecules Too many peaks overlapping one another to easily interpret Difficult to extract peak intensity information Peaks are broad and generally lose all the fine details (J-coupling) Is there a way of spreading out all these peaks? (Yes! 2D NMR)

40. 2D Gels & 2D NMR* SDS PAGE

41. Multidimensional NMR* 1D 2D 3D MW ~ 500 MW ~ 10,000 MW ~ 30,000

42. The NMR Process* Obtain protein sequence Collect TOCSY & NOESY data Use chemical shift tables and known sequence to assign TOCSY spectrum Use TOCSY to assign NOESY spectrum Obtain inter and intra-residue distance information from NOESY data Feed data to computer to solve structure

43. Multidimensional NMR NOESY TOCSY

44. white Assigning Chemical Shifts TOCSY (white) NOESY (red)

45. The NOE* NOEs build up over time (50-400 ms) NOEs are stronger for 1 H atoms that are closer together in space NOEs are weaker for 1 H atoms far away from each other Limit is from 1.5-5.5 Angstroms

46. Measuring NOEs*

47. NMR Spectroscopy* Chemical Shift Assignments NOE Intensities J-Couplings Distance Geometry Simulated Annealing

48. NMR Spectroscopy & Protein Structure NMR generates multiple structures (called an ensemble) instead of just one structures (as in X-ray) This reflects the fact that there are multiple “solutions” to the set of distance (NOE, J-coupling, Hbond) restraints provided to the computer The more “blurred” the region or loop, the more flexible it likely is

49. The Final Result ORIGX2 0.000000 1.000000 0.000000 0.00000 2TRX 147 ORIGX3 0.000000 0.000000 1.000000 0.00000 2TRX 148 SCALE1 0.011173 0.000000 0.004858 0.00000 2TRX 149 SCALE2 0.000000 0.019585 0.000000 0.00000 2TRX 150 SCALE3 0.000000 0.000000 0.018039 0.00000 2TRX 151 ATOM 1 N SER A 1 21.389 25.406 -4.628 1.00 23.22 2TRX 152 ATOM 2 CA SER A 1 21.628 26.691 -3.983 1.00 24.42 2TRX 153 ATOM 3 C SER A 1 20.937 26.944 -2.679 1.00 24.21 2TRX 154 ATOM 4 O SER A 1 21.072 28.079 -2.093 1.00 24.97 2TRX 155 ATOM 5 CB SER A 1 21.117 27.770 -5.002 1.00 28.27 2TRX 156 ATOM 6 OG SER A 1 22.276 27.925 -5.861 1.00 32.61 2TRX 157 ATOM 7 N ASP A 2 20.173 26.028 -2.163 1.00 21.39 2TRX 158 ATOM 8 CA ASP A 2 19.395 26.125 -0.949 1.00 21.57 2TRX 159 ATOM 9 C ASP A 2 20.264 26.214 0.297 1.00 20.89 2TRX 160 ATOM 10 O ASP A 2 19.760 26.575 1.371 1.00 21.49 2TRX 161 ATOM 11 CB ASP A 2 18.439 24.914 -0.856 1.00 22.14 2TRX 162

50. How Long Does it Take? Chemical Shift Assignments NOE Intensities J-Couplings Distance Geometry MD Refinement 6-12 months2-4 months1-2 months Evaluation

51. High Throughput NMR Higher magnetic fields (From 400 MHz to 900 MHz) Higher dimensionality (From 2D to 3D to 4D) New pulse sequences (TROSY, CBCA(CO)NH) Improved sensitivity New parameters (Dipolar coupling, cross relaxation)

52. Automated NMR Structure Generation

53. X-ray Versus NMR (Bottlenecks)* Producing enough protein for trials Crystallization time and effort Crystal quality, stability and size control Finding isomorphous derivatives Chain tracing & checking Producing enough labeled protein for collection Sample “conditioning” Size of protein Assignment process is slow and error prone Measuring NOE’s is slow and error prone X-rayNMR

54. Conclusions Approximately ¼ of all structure in the PDB have been generated by NMR Proteins as large as 700 residues have been analyzed/solved by NMR Excellent method for studying protein structure in solution, probing dynamics, flexibility, stability, kinetics and rate processes NMR compliments X-ray work