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

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 Blanchet

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

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

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

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

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

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

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

“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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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