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  1. NMR SpectroscopyQuestion and Answer Session Judith Klein-SeetharamanDepartment of Structural Biology jks33@pitt.edu

  2. What did we do? • NMR instrumentation • NMR sample preparation considerations • Water signals • Water suppression • Detergents • NMR setup – downstairs • zg • zgpr • selabs • hsqc • Spectral processing: Topspin Software Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  3. What did we do then? • Converting raw spectral formats to other formats using NMRpipe • Example: HIV protease relaxation data • Assignment of signals with one strategy • HNCO • HNCOCACBG • HNCOCA • HNCACB • HNCA • HNCACO • Example: Ubiquitin assignment with NMRviewJ Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  4. What would be the next step? • For determining a structure with NMR spectroscopy, after assignment, we would… • Collect constraints: • NOE • Dipolar coupling • Scalar coupling constants (gives dihedral angles) • Solvent exchange • Calculate a structure based on these constraints Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  5. What are other things one can do with NMR? • For study of dynamics • Measure and analyze T1, T2, HetNOE data • We went through T1 analysis in computer lab (conversion of raw data with NMRpipe, analysis with NMRviewJ) • T2 was your homework • Other studies might include chemical shift perturbations • Temperature titrations • … Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  6. Homework Assignments Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  7. Homework Part 1 • NMR instrumentation • NMR sample preparation considerations • Water signals • Water suppression • Detergents • NMR setup – downstairs • zg • zgpr • selabs • hsqc • Spectral processing: Topspin Software Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  8. Experiments Run • zg = records all 1H's in your sample • zgpr = same as zg but with water suppression • selabs = the way we set it up, it records only NH region • hsqcfpgpf3gphwg = in short: hsqc = records all 1H's attached to 15N Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  9. click on Topspin icon and open the program Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  10. click on guest, expand MB3_Jan2007 directory • This is the directory that contains all of the data that was acquired in class. Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  11. Right click on MB3_Jan2007 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  12. Select Expand & Show PULPROG/Title Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  13. Samples • Sample IDs Experiment IDs • B 1-7 • F 10-15 • A 20-23 • D 30-33 • E 40-43 • H 50-53 • B 60, 61 • F 70, 71 • F 100-103 • B 110-113 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  14. 40 = zg of sample E • 41 = zgpr of sample E • 42 = selabs of Sample E • 43 = hsqc of Sample E • 50 = zg of Sample H • 51 = zgpr of Sample H • 52 = selabs of sample H • 53 = hsqc of sample H • 60 = zg of sample B • 61 = zgpr of sample B • 70 = zg of sample F • 71 = zgpr of sample F • -----------------experiments done with comp bio students • 100 = zg of sample F • 101 = zgpr of sample F • 102 = selabs of sample F • 103 = hsqc of sample F • 110 = zg of sample B • 111 = zg of sample B • 112 = selabs of sample B • 113 = hsqc of sample B Samples • 1 = zgpr of Sample B • 2 = zgpr of Sample B • 3 = hsqc of Sample B • 4 = zg of Sample B • 5 = zg of Sample B • 6 = selabs of Sample B • 7 = selabs of Sample B • 10 = zg of Sample F • 11 = selabs of Sample F • 12 = zgpr of Sample F • 13 = hsqc of sample F • 14 = hsqc of sample F • 15 = hsqc of sample F • ----- experiments done with MB3 students: • 20 = zgpr of Sample A • 21 = zg of sample A • 22 = selabs of sample A • 23 = hsqc of sample A • 30 = zg of Sample D • 31 = zgpr of Sample D • 32 = selabs of Sample D • 33 = hsqc of Sample D Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  15. Solving the Mystery of the Samples • Drag the experiment that you want to analyze into the main purple window area. • If you do not see a spectrum, you need to type efp • This will process the data so that you can see the spectrum. • In the case of the hsqc it will also ask you for an experiment number where to store the processed data, you can use the default or enter a number, • e.g. 2. Another alternative is to type “rser 1” that will retrieve the first slice of the 2d hsqc experiment also. Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  16. To see what acquisition parameters were chosen, click on the AcquPars tab, you will see a window: Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  17. Notice that you can scroll down to see many fields under this tab. • If you type ased only those parameters relevant to the specific experiment are shown: Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  18. Some tips • If you want to move more quickly from experiment to experiment, type re and then the experiment number, e.g. • re 21 gives the experiment stored in directory 21 • To save a picture of a spectrum select File, Export and then give it a file name extension indicating which format you would like (e.g. .jpg) • Note that you may have to phase your spectrum. Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  19. Phasing Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  20. Sample A. 20-23 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  21. Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  22. Overlay Sample A • From these spectra, we can conclude that we do not have 15N label, it i a water sample. It does not have detergent in it, or the detergent is deuterated. 22 = selabs 21 = zg 20 = zgpr 23 = hsqc Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  23. Sample B. 1-7, 60-61, 110-113 3 = hsqc 8 = selabs 4 = zg 2 = zgpr Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  24. Sample D. 30-33 33 = hsqc 32 = selabs 31 = zgpr 30 = zg Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  25. Sample E. 40-43 42 = selabs 41 = zgpr 40 = zg 43 = hsqc Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  26. Sample F. 10-15, 70-71, 100-103 103 = hsqc 102 = selabs 101 = zgpr 100 = zg Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  27. Sample H. 50-53 52 = selabs – disregard this spectrum 53 = hsqc 51 = zgpr 50 = zg Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  28. Parameters to watch out for • ns • rg • will affect actual intensity measured. Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  29. Several different assignment strategies exist Most easily automated: • HNCO • HNCOCACB • HNCOCA • HNCACB • HNCA • HNCACO Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  30. Homework Part 2 • Converting raw spectral formats to other formats using NMRpipe • Example: HIV protease relaxation data • Assignment of signals with one strategy • HNCO • HNCOCACBG • HNCOCA • HNCACB • HNCA • HNCACO • Example: Ubiquitin assignment with NMRviewJ Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  31. This is a cluster: Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  32. HNCO Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  33. HNCA Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  34. HNCACO Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  35. HNCOCACB, similar: CBCA(CO)NH Not in HNCOCACB experiment Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  36. HNCOCA Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  37. HNCACB Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  38. What are other things one can do with NMR? • For study of dynamics • Measure and analyze T1, T2, HetNOE data • We went through T1 analysis in computer lab (conversion of raw data with NMRpipe, analysis with NMRviewJ) • T2 was your homework • Other studies might include chemical shift perturbations • Temperature titrations • … Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  39. Dynamics in folded/unfolded lysozyme Unfolded: Folded: Smaller rates – more flexible Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  40. T1 for HIV protease Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  41. T2 for HIV protease Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  42. T2 for HIV protease Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  43. From last class in case there are questions Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  44. NMR parameters Chemical Shift H2O methyl aromatic Trp-side-chain NH OH aliphatic Backbone NH Side-chain HN Ha Spectrum see handout Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  45. NMR of membrane proteins In detergent micelle: In lipid bilayer: http://www.elmhurst.edu/~chm/vchembook/558micelle.html Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  46. Problems! Detergent peaks Detergent signals cause dynamic range problems (Detergent signals cause spectral overlap) Detergent deuteration is often not feasible Problem: 1H,1H NOESY spectra do not show protein signals Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  47. Selective excitation B. Selective excitation of the same region as in A. Using excitation sculpting. A. Selective excitation of the NH region using 90 degree pulse followed by direct observation. Backbone NH Tryptophan side chain NH 20 15 10 5 10 5 0 -5 1H Chemical Shift [ppm] 1H Chemical Shift [ppm] Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  48. 2d HSQC Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session

  49. 1d projection of HSQC Computational Biology Laboratory Course – Klein-Seetharaman – NMR Question Answer Session