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NMR Spectroscopy

NMR Spectroscopy. Judith Klein-Seetharaman Department of Structural Biology jks33@pitt.edu. Objectives of this Lecture and Practicum. Resources Physical principle Sample requirements Parameters that are measured by NMR Dynamics by NMR Limitations Practical aspects

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NMR Spectroscopy

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  1. NMR Spectroscopy Judith Klein-SeetharamanDepartment of Structural Biology jks33@pitt.edu

  2. Objectives of this Lecture and Practicum • Resources • Physical principle • Sample requirements • Parameters that are measured by NMR • Dynamics by NMR • Limitations • Practical aspects • Setup of NMR experiments (downstairs) Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  3. Resources Websites • http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf • http://www.bmrb.wisc.edu/ • http://www.biochem.ucl.ac.uk/bsm/nmr/ubq/ • http://nobelprize.org/nobel_prizes/chemistry/laureates/2002/wutrich-lecture.pdf • http://www.cis.rit.edu/htbooks/nmr/ • http://www.ch.ic.ac.uk/local/organic/nmr.html • http://www.spectroscopynow.com/ • http://www.chem.queensu.ca/FACILITIES/NMR/nmr/webcourse/ • http://spincore.com/nmrinfo/ • http://www.chembio.uoguelph.ca/driguana/NMR/TOC.HTM • http://www.embl-heidelberg.de/nmr/sattler/embo/handouts/griesinger_lecture_pof.pdf • http://dupont.molbio.ku.dk/teach/course/introNMR.pdf • http://www.infochembio.ethz.ch/links/en/spectrosc_nmr_lehr.html Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  4. Resources Books NMR Books: • Protein NMR Techniques (Methods in Molecular Biology) by A. Kristina Downing (Editor) • Protein NMR Spectroscopy: Principles and Practice by John Cavanagh, Wayne J. Fairbrother, III, Arthur G. Palmer, Nicholas J. Skelton, Mark Rance • Spin Dynamics: Basics of Nuclear Magnetic Resonance by Malcolm H. Levitt • Principles of Nuclear Magnetic Resonance in One and Two Dimensions by Richard R. Ernst, Geoffrey Bodenhausen, Alexander Wokaun • 200 and More NMR Experiments: A Practical Course by Stefan Berger, Siegmar Braun • Basic One- and Two-Dimensional NMR Spectroscopy by Horst Friebolin • NMR Spectroscopy: Basic Principles, Concepts, and Applications in Chemistry by Harald Günther • NMR Data Processing by Hoch • NMR: The Toolkit by P. J. Hore, J. A. Jones, S. Wimperis • Nuclear Magnetic Resonance by P. J. Hore • NMR for Physical and Biological Scientists by Thoma Pochapsky • Understanding NMR Spectroscopy by James Keeler • NMR of Proteins (Topics on Molecular and Structural Biology) by G. M. Clore, A. M. Gronenborn • The Nuclear Overhauser Effect in Structural and Conformational Analysis by David Neuhaus, Michael P. Williamson Biophysics Books with chapters on NMR: • Biophysical Chemistry: Part II: Techniques for the Study of Biological Structure and Function by Charles R Cantor, Paul R Schimmel • Principles of Physical Biochemistry by Kensal E van Holde, Curtis Johnson, Pui Shing Ho Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  5. Objectives of this Lecture and Practicum • Resources • Physical principle • Sample requirements • Parameters that are measured by NMR • Dynamics by NMR • Limitations • Practical aspects • Setup of NMR experiments (downstairs) Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  6. Nuclei in a magnetic field http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  7. Energy Difference http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  8. Macroscopic View http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  9. http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  10. Experiment: Recycle delay dependent on T1 relaxation http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  11. The NMR signal http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf • Analogy: conducting loop rotating in a magnetic field: Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  12. Fourier Transform http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  13. Soft pulses vs. hard pulses http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  14. Obtaining a spectrum http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  15. Product Operator Formalism http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  16. http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  17. http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  18. HSQC Experiment http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  19. HSQC TOCXY http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  20. Signal Intensity http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf • Boltzmann distribution Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  21. Objectives of this Lecture and Practicum • Resources • Physical principle • Sample requirements • Parameters that are measured by NMR • Dynamics by NMR • Limitations • Practical aspects • Setup of NMR experiments (downstairs) Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  22. Sample requirements: Sources Think of the requirements that we may need to fulfil! Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  23. Example Comparison of expression systems for rhodopsin http://www.gla.ac.uk/ibls/BMB/mdh/images/conrd1-cos-golgi.gif http://www.wjgnet.com/images/english/V11/2576-2a.jpg http://www.icr.ac.uk/structbi/baculovirus/img/infectedsf9.jpg spacebio.net/modules/ mb_teare.html genetics.med.harvard.edu/ ~winston/ ___________ ___________ ___________ ___________ ___________ ___________ What are the advantages and disadvantages of each expression system? Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  24. Where can we get these molecules from? Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  25. Sources of biomolecules Summary • Native sources • Best quality (correct fold, posttranslational modifications etc.) • Not always best quantity • Limitations in labeling • No mutants • Chemical synthesis • Good for small molecules • Not good for large proteins • Biosynthesis • A variety of expression systems exist, all with their advantages and disadvantages. • Required for isotope labeling for NMR Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  26. Objectives of this Lecture and Practicum • Resources • Physical principle • Sample requirements • Parameters that are measured by NMR • Dynamics by NMR • Limitations • Practical aspects • Setup of NMR experiments (downstairs) Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  27. NMR parameters Chemical Shift http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf See handout Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  28. Chemical shift perturbation Figure 2 in “Cap-free structure of eIF4E suggests a basis for conformational regulation by its ligands Laurent Volpon, Michael J Osborne, Ivan Topisirovic, Nadeem Siddiqui and Katherine LB Borden The EMBO Journal (2006) 25, 5138–5149 Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  29. NMR parameters The Nuclear Overhauser Effect http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  30. Measuring NOE’s http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  31. NMR Parameters Dipolar Couplings http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  32. NMR Structure of Bcl-XL Bound to BH3 Peptide • Structure was solved with a homolog of BH3 helix. • Protein-protein interaction groove was identified on anti-apoptotic Bcl-XL. Identify a drug that binds in the BH3 pocket of Bcl-XL, inhibit binding to BID -> normal apoptosis. PDB ID:1G5J Drug design approach: SAR by NMR Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  33. NMR parameters • chemical shifts • NOE • Dipolar coupling • coupling constants • HetNOE • longitudinal relaxation rates (R1) • transverse relaxation rates (R2) Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  34. Objectives of this Lecture and Practicum • Resources • Physical principle • Sample requirements • Parameters that are measured by NMR • Dynamics by NMR • Limitations • Practical aspects • Setup of NMR experiments (downstairs) Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  35. Theory-NMR Relaxation mechanism NMR Dynamics on Different Time Scales time scaletype ns-ps fast internal motions us-ms slow internal motions ms-days proton exchange Protein are dynamic molecules http://www.bioc.aecom.yu.edu/labs/girvlab/nmr/course/relaxdyn NMR dynamics can be used on a broad range of timescales. Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  36. Relaxation • Longitudinal relaxation (T1): return of longitudinal (z-component) to its equilibrium value • Transverse relaxation (T2): decay of transverse (x,y-component) Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  37. T1 Relaxation http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  38. Experiment: Recycle delay dependent on T1 relaxation http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  39. T2 Relaxation http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  40. Mechanisms of Relaxation http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf • Dipolar interaction Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  41. Mechanisms of Relaxation http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf • Chemical shift anisotropy • Scalar relaxation (chemical exchange, rapid T1 relaxation) • Quadrupolar relaxation • Spin rotation relaxation • Interaction with unpaired electrons Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  42. Quantification of motion- strategies to obtain dynamic information from NMR relaxation experiment Measure R1, R2, heteronuclear NOE “model free” approach Get order parameter S2 ,τe, τm Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  43. Lipari-Szabo Model Free Approach http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  44. Lipari Szabo http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf • Order parameters S2 • τe, effective correlation function time for internal motions • τm, overall tumbling correlation time for global motions Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  45. Lipari–Szabo “model-free” approach- don’t depend on a specific physical model • Estimate (τm) from R2/R1 for a selected subset of the residues • fits to the observed relaxation data using various regression variables • model-selection criteria are used to decide which choice is appropriate for each residue • Reoptimize using the selected models. • Uncertainties in the optimized parameters were obtained by Monte Carlo simulation. Michael Andrec, Gaetano T. Montelione, RonaldM. Levy Journal of Magnetic Resonance 139, 408–421 (1999) “Model-free” is the most popular method to calculate dynamic parameters Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  46. Inversion Recovery: Measure T1 http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  47. Carr-Purcell spin echo: Measure T2 http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  48. Comparison of T1 and T2 relaxation http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  49. Dependence of T1, T2 on Tumbling Time http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

  50. Chemical Exchange http://www.oci.unizh.ch/group.pages/zerbe/NMR.pdf Computational Biology Laboratory Course – Klein-Seetharaman – NMR Lecture

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