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Time resolved scattering studies Clement Blanchet Time resolved study - PowerPoint PPT Presentation

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Time resolved scattering studies Clement Blanchet Time resolved study Collect data at different time point to study sample whose structure are evolving in time A tool to study kinetics Time resolved scattering studies C. 12/4/2012

  1. Time resolved scattering studies Clement Blanchet

  2. Time resolved study • Collect data at different time point to study sample whose structure are evolving in time • A tool to study kinetics Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  3. Kinetic experiment • Perturb a system • Monitor the return to equilibrium Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  4. Perturbation • Different techniques: – Mixing – T, P jump – Light triggered reaction, …. • Homogeneous perturbation • Fast perturbation for fast kinetic Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  5. Monitor the reaction • Fast reaction � short collection time • But one need enough photons to collect a proper SAXS data � High flux Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  6. High flux • Third generation synchrotron 1e-6 1e-6 1e-6 • Multilayer monochromator 8e-7 8e-7 8e-7 6e-7 6e-7 6e-7 4e-7 4e-7 4e-7 2e-7 2e-7 2e-7 0 0 • Pink beam 0 -2e-7 -2e-7 -2e-7 11600 11600 11800 11800 12000 12000 12200 12200 12400 12400 12600 12600 12800 12800 13000 13000 11600 11800 12000 12200 12400 12600 12800 13000 Undulator Double crystal monochromator Time resolved scattering studies ‐ C. 12/4/2012 Multilayer monochromator Blanchet

  7. Dead time • Time between the beginning of the reaction and the first data point • Depends on: – How fast the reaction is triggered – How fast the first point can be collected • Short dead time needed to study fast kinetic Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  8. Time scale of biological processes Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  9. Examples • “Slow Kinetics” – Fibril formation • Sub ‐ Second kinetics – Stopped ‐ flow • Millisecond kinetics – Continuous flow – Caged compound • Ultrafast kinetics Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  10. “slow” kinetics Vestergaard, B., Groenning, M., Roessle, M., Kastrup, J.S., de Weert, M.V., Flink, J.M., Frokjaer, S., Gajhede, M. & Svergun,D.I. (2007) PLoS Biol. 5, e134 Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  11. Subsecond kinetics • Stopped ‐ flow (dead time: 1 ‐ 10 ms) Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  12. Stopped flow ‐ Example Characterization of Transient Intermediates in Lysozyme Folding with Time ‐ resolved Small ‐ angle X ‐ ray Scattering Segel et al. JMB , 1999, Volume 288 (3), 489 ‐ 499 Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  13. Lysozyme Folding Lysozyme 1 x 3.6M GdmCl Lysozyme 0.6M GdmCl Buffer 5 x Without GdmCl Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  14. Lysozyme Folding • Evolution of Rg in time Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  15. Singular value decomposition (Wildegger & Kiefhaber, 1997) Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  16. Interrupted refolding experiment • Double mixing step monitored by fluorescence Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  17. Reconstruction of the scattering profile = ν + ν + ν I ( s , t ) ( t ) I ( s ) ( t ) I ( s ) ( t ) I ( s ) C C I I N N ∑ = I ( s ) v I ( s ) k k k Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  18. Continuous flow Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  19. Continuous flow • Continuous flow � high sample consumption – Microfluidic continuous flow system • Space < ‐ > time – low flux OK – time resolution < ‐ > flow rate and size of the beam • Dead time ≈ 150 microseconds Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  20. Example continuous flow Conformational landscape of cytochrome c folding studied by microsecond ‐ resolved small ‐ angle x ‐ ray scattering. Akiyama et al. PNAS 2002 Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  21. Continuous flow Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  22. Radius of gyration Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  23. Kratky plots Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  24. Singular value decomposition Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  25. Conformational landscape of Cyto C Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  26. Caged compound release by flash photolysis • DM ‐ nitrophen Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  27. Calmodulin A Compact Intermediate State of Calmodulin in the Process of Target Binding. Yamada et al. Biochemistry 2012 Mastoparan Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  28. Equilibrium measurement Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  29. Kinetics Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  30. 140 ms 10 ms 0.5 ms 30 s With mastoparan Without mastoparan Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  31. Model Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  32. Ultra ‐ fast time resolved Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  33. Ultra short collection time • Beamline ID09B, ESRF, Grenoble • Using the pulsed structure of the synchrotron • About 5000000 bunch/sec Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  34. Isolate one bunch • Isolate one bunch (ms shutter + fast chopper) Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  35. Single bunch experiment • High flux needed • Repetition of the measurements Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  36. Pump and probe experiment Trigger with Probe with Laser pulse X ‐ ray τ t Bunch length ≈ 100 ps � Resolution: up to 100 ps Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  37. What is 100ps 100 psec � second � 315 years Second Louis XIV Light travels 3 cm in 100ps Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  38. Too fast for SAXS Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  39. TR WAXS Tracking the structural dynamics of proteins in solution using time ‐ resolved wide ‐ angle X ‐ ray scattering. Cammarata et al. Nature 2008. Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  40. T and R states of hemoglobin Looking at the unbinding of oxygen by hemoglobin Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  41. Experimental setup Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  42. Structural change in hemoglobin Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

  43. Conclusion • SAS can be used to study kinetic • For fast reaction: – Special setup required to triggered the reaction – High flux is needed: third generation source (impossible with lab source and neutrons) Time resolved scattering studies ‐ C. 12/4/2012 Blanchet

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