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

▶
time resolved scattering studies
SMART_READER_LITE
LIVE PREVIEW

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

Mar 09, 2024 38 likes •470 views

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

slide-1
SLIDE 1

Time resolved scattering studies

Clement Blanchet

slide-2
SLIDE 2

Time resolved study

  • Collect data at different time point to study

sample whose structure are evolving in time

  • A tool to study kinetics

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

slide-3
SLIDE 3

Kinetic experiment

  • Perturb a system
  • Monitor the return to equilibrium

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

slide-4
SLIDE 4

Perturbation

  • Different techniques:

– Mixing – T, P jump – Light triggered reaction, ….

  • Homogeneous perturbation
  • Fast perturbation for fast kinetic

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

slide-5
SLIDE 5

Monitor the reaction

  • Fast reaction short collection time
  • But one need enough photons to collect a

proper SAXS data High flux

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

slide-6
SLIDE 6 11600 11800 12000 12200 12400 12600 12800 13000
  • 2e-7
2e-7 4e-7 6e-7 8e-7 1e-6 11600 11800 12000 12200 12400 12600 12800 13000
  • 2e-7
2e-7 4e-7 6e-7 8e-7 1e-6 11600 11800 12000 12200 12400 12600 12800 13000
  • 2e-7
2e-7 4e-7 6e-7 8e-7 1e-6

High flux

  • Third generation synchrotron
  • Multilayer monochromator
  • Pink beam

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

Undulator Double crystal monochromator Multilayer monochromator

slide-7
SLIDE 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

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

slide-8
SLIDE 8

Time scale of biological processes

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

slide-9
SLIDE 9

Examples

  • “Slow Kinetics”

– Fibril formation

  • Sub‐Second kinetics

– Stopped‐flow

  • Millisecond kinetics

– Continuous flow – Caged compound

  • Ultrafast kinetics

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

slide-10
SLIDE 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

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

slide-11
SLIDE 11

Subsecond kinetics

  • Stopped‐flow (dead time: 1‐10 ms)

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

slide-12
SLIDE 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

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

slide-13
SLIDE 13

Lysozyme Folding

Lysozyme 3.6M GdmCl Buffer Without GdmCl Lysozyme 0.6M GdmCl 1 x 5 x

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

slide-14
SLIDE 14

Lysozyme Folding

  • Evolution of Rg in time

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

slide-15
SLIDE 15

Singular value decomposition

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

(Wildegger & Kiefhaber, 1997)

slide-16
SLIDE 16

Interrupted refolding experiment

  • Double mixing step monitored by fluorescence

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

slide-17
SLIDE 17

Reconstruction of the scattering profile

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

) ( ) ( ) ( ) ( ) ( ) ( ) , ( s I t s I t s I t t s I

N N I I C C

ν ν ν + + =

∑

=

k k k

s I v s I ) ( ) (

slide-18
SLIDE 18

Continuous flow

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

slide-19
SLIDE 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

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

slide-20
SLIDE 20

Example continuous flow

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

Conformational landscape of cytochrome c folding studied by microsecond‐resolved small‐angle x‐ray scattering. Akiyama et al. PNAS 2002

slide-21
SLIDE 21

Continuous flow

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

slide-22
SLIDE 22

Radius of gyration

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

slide-23
SLIDE 23

Kratky plots

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

slide-24
SLIDE 24

Singular value decomposition

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

slide-25
SLIDE 25

Conformational landscape of Cyto C

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

slide-26
SLIDE 26

Caged compound release by flash photolysis

  • DM‐nitrophen

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

slide-27
SLIDE 27

Calmodulin

A Compact Intermediate State of Calmodulin in the Process of Target

  • Binding. Yamada et al. Biochemistry 2012

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

Mastoparan

slide-28
SLIDE 28

Equilibrium measurement

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

slide-29
SLIDE 29

Kinetics

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

slide-30
SLIDE 30

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

0.5 ms 10 ms 140 ms 30 s With mastoparan Without mastoparan

slide-31
SLIDE 31

Model

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

slide-32
SLIDE 32

Ultra‐fast time resolved

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

slide-33
SLIDE 33

Ultra short collection time

  • Beamline ID09B, ESRF, Grenoble
  • Using the pulsed structure of the synchrotron
  • About 5000000 bunch/sec

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

slide-34
SLIDE 34

Isolate one bunch

  • Isolate one bunch (ms shutter + fast chopper)

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

slide-35
SLIDE 35

Single bunch experiment

  • High flux needed
  • Repetition of the measurements

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

slide-36
SLIDE 36

Pump and probe experiment

t

Trigger with Laser pulse Probe with X‐ray τ

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

Bunch length ≈ 100 ps Resolution: up to 100 ps

slide-37
SLIDE 37

What is 100ps

100 psec second Second 315 years Light travels 3 cm in 100ps

Louis XIV

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

slide-38
SLIDE 38

Too fast for SAXS

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

slide-39
SLIDE 39

TR WAXS

Tracking the structural dynamics of proteins in solution using time‐resolved wide‐angle X‐ray scattering. Cammarata et al. Nature 2008.

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

slide-40
SLIDE 40

T and R states of hemoglobin

Looking at the unbinding of oxygen by hemoglobin

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

slide-41
SLIDE 41

Experimental setup

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

slide-42
SLIDE 42

Structural change in hemoglobin

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

slide-43
SLIDE 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)

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