Time resolved scattering experiments Clement Blanchet Time - - PowerPoint PPT Presentation

▶
time resolved scattering experiments
SMART_READER_LITE
LIVE PREVIEW

Time resolved scattering experiments Clement Blanchet Time - - PowerPoint PPT Presentation

Jun 20, 2023 456 likes •1.06k views

Time resolved scattering experiments Clement Blanchet Time resolved experiments? Studies of systems that changes over time Collect data at different time point of the reaction Time resolved scattering studies - C. 6/24/2016 Blanchet

slide-1
SLIDE 1

Time resolved scattering experiments

Clement Blanchet

slide-2
SLIDE 2

Time resolved experiments?

  • Studies of systems that changes over time
  • Collect data at different time point of the

reaction

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-3
SLIDE 3

Time scale of biological processes

(protein folding)

6/24/2016 Time resolved scattering studies - C. Blanchet

Different time scales Different experimental setups

slide-4
SLIDE 4

Ingredients of time resolved experiments

  • Controlled triggering of the reaction of

interest

  • Way of monitoring the reaction

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-5
SLIDE 5

Triggering

6/24/2016 Time resolved scattering studies - C. Blanchet

Modification of physical condition (T, P) Modification of chemical conditions

  • pH jump
  • Denaturing agent
  • Addition of salts or other additives

Modification of the system itself Flash photolysis, photosensitive protein

slide-6
SLIDE 6

Limitation – triggering

  • Triggering:

– Simultaneous, fast and homogeneous triggering at the time scale of the reaction

6/24/2016 Time resolved scattering studies - C. Blanchet

0.2 0.4 0.6 0.8 1 1.2 50 100 150 200 0.2 0.4 0.6 0.8 1 1.2 50 100 150 200 0.2 0.4 0.6 0.8 1 1.2 50 100 150 200

slide-7
SLIDE 7

How fast can you trigger the reaction?

  • Depends on the triggering methods

– Mixing:

  • seconds to ms (with fast mixing devices)
  • Limited by mixing, diffusion time

– P-Jump:

  • Diffusion of the pressure shockwave: speed of sound ms
  • In practice micros-ms

– Light triggered reaction:

  • Practically not limited for “direct” triggering (limitation:

speed of light)

  • Limited by intermediate reaction in the case of indirect

triggering (T-Jump, caged compound)

6/24/2016 Time resolved scattering studies - C. Blanchet

* Small measurement cell helps.

slide-8
SLIDE 8

Monitoring the reaction

  • Many spectroscopic technics can and have been used
  • SAXS is a good technics to study reaction of biological

system

– Samples are in solution, in a quasi-native state. Many reaction takes place in solution and can be triggered in a controlled manner – Data can be collected quickly: Possibility to study fast reaction

  • SANS: long collection time, limited to very slow reaction
  • Different mode of data collection

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-9
SLIDE 9

Continuous vs pump-probe

6/24/2016 Time resolved scattering studies - C. Blanchet

t t

Perturbation Perturbation Probe Probe

∆t ∆t

Continuous Pump-probe

slide-10
SLIDE 10

Continuous vs pump-probe

6/24/2016 Time resolved scattering studies - C. Blanchet

t t

Perturbation Perturbation Probe Probe

∆t ∆t

slide-11
SLIDE 11

Continuous vs pump-probe

6/24/2016 Time resolved scattering studies - C. Blanchet

t t

Perturbation Perturbation Probe Probe

∆t ∆t

slide-12
SLIDE 12

Limitation – Collection time

6/24/2016 Time resolved scattering studies - C. Blanchet

0.2 0.4 0.6 0.8 1 50 100 150 200 0.2 0.4 0.6 0.8 1 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 0.2 0.4 0.6 0.8 1 50 100 150 200

slide-13
SLIDE 13

Limitation – Collection time

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-14
SLIDE 14

Short collection time - Fast detector

  • Photon counting detector: Pilatus (300Hz),

Eiger (up to 3kHz), Xfel detectors,…

  • Gas detector (Theoretically, up to 1MHz)

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-15
SLIDE 15

Short collection time – Short X-ray pulse

  • Use short beam pulse to overcome the

detector limitation (using fast shutter, chopper,…)

6/24/2016 Time resolved scattering studies - C. Blanchet

Chopper

Detector collection X-ray pulse

slide-16
SLIDE 16 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

Short collection time: High flux

  • Third generation synchrotron
  • Multilayer monochromator
  • Pink beam

6/24/2016 Time resolved scattering studies - C. Blanchet

Undulator Double crystal monochromator Multilayer monochromator

slide-17
SLIDE 17

Example high flux beam (BL40XU, Spring8)

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-18
SLIDE 18

But careful radiation damage

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-19
SLIDE 19

But careful radiation damage

  • Adapt collection strategy (pump and probe)
  • Use short pulses

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-20
SLIDE 20

FEL Beam (SLAC Stanford)

6/24/2016 Time resolved scattering studies - C. Blanchet

Stan, C. A., Milathianaki, D., Laksmono, H., Sierra, R. G., McQueen, T. A., Messerschmidt, M., ... & Guillet, S. A. (2016). Liquid explosions induced by X-ray laser pulses. Nature Physics.

slide-21
SLIDE 21

Dead time

  • Time between the reaction is triggered and

the first point is collected (depends on triggering methods and collection time)

6/24/2016 Time resolved scattering studies - C. Blanchet

0.2 0.4 0.6 0.8 1 50 100 150 200

Short dead time required to study fast kinetic

slide-22
SLIDE 22

Examples

  • Sub-Second TR experiments

– Stopped-flow

  • Millisecond TR experiments

– Continuous flow – Caged compound

  • Ultrafast TR experiments

– Synchrotron – FEL

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-23
SLIDE 23

Sub-second kinetics

  • Stopped-flow (dead time: 2-10 ms)

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-24
SLIDE 24

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

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-25
SLIDE 25

Lysozyme Folding

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

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-26
SLIDE 26

Lysozyme Folding

  • Evolution of Rg in time

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-27
SLIDE 27

Refolding model

6/24/2016 Time resolved scattering studies - C. Blanchet

(Wildegger & Kiefhaber, 1997)

U C

Rg = 23.5 A Rg = 19.6 A

slide-28
SLIDE 28

Interrupted refolding experiment

  • Double mixing step monitored by fluorescence

6/24/2016 Time resolved scattering studies - C. Blanchet

C

slide-29
SLIDE 29

Interrupted refolding experiment

  • Double mixing step monitored by fluorescence

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-30
SLIDE 30

Reconstruction of the scattering profile

6/24/2016 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-31
SLIDE 31

Refolding model

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-32
SLIDE 32

Continuous flow

6/24/2016 Time resolved scattering studies - C. Blanchet

Concentric capillary mixer Mixing time: 30 microseconds

Moskowitz & Bowman, Science, 1966

Turbulent mixing Laminar mixing

slide-33
SLIDE 33

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 (SAXS) ≈150 microseconds

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-34
SLIDE 34

Example continuous flow

6/24/2016 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-35
SLIDE 35

Continuous flow

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-36
SLIDE 36

Radius of gyration

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-37
SLIDE 37

Kratky plots

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-38
SLIDE 38

SAXS Curves

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-39
SLIDE 39

Conformational landscape of Cyto C

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-40
SLIDE 40

Caged compound release by flash photolysis

  • DM-nitrophen

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-41
SLIDE 41

Calmodulin

A Compact Intermediate State of Calmodulin in the Process of Target

  • Binding. Yamada et al. Biochemistry 2012

6/24/2016 Time resolved scattering studies - C. Blanchet

Mastoparan

slide-42
SLIDE 42

Equilibrium measurement

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-43
SLIDE 43

Kinetics

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-44
SLIDE 44

6/24/2016 Time resolved scattering studies - C. Blanchet

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

slide-45
SLIDE 45

Model

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-46
SLIDE 46

ULTRA-FAST TIME RESOLVED

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-47
SLIDE 47

Ultra short collection time

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

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-48
SLIDE 48

Isolate one bunch

  • Isolate one bunch (ms shutter + fast chopper)

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-49
SLIDE 49

Single bunch experiment

  • High flux needed
  • Repetition of the measurements

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-50
SLIDE 50

Pump and probe experiment

t

Trigger with Laser pulse Probe with X-ray τ

6/24/2016 Time resolved scattering studies - C. Blanchet

Bunch length ≈ 100 ps Resolution: up to 100 ps

slide-51
SLIDE 51

What is 100ps?

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

6/24/2016 Time resolved scattering studies - C. Blanchet

? ?

slide-52
SLIDE 52

TR WAXS

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

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-53
SLIDE 53

T and R states of hemoglobin

Looking at the unbinding of oxygen by hemoglobin

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-54
SLIDE 54

Experimental setup

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-55
SLIDE 55

Structural change in hemoglobin

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-56
SLIDE 56

FEL

6/24/2016 Time resolved scattering studies - C. Blanchet

Levantino, M., Schirò, G., Lemke, H. T., Cottone, G., Glownia, J. M., Zhu, D., ... & Cammarata, M. (2015). Ultrafast myoglobin structural dynamics observed with an X-ray free-electron

  • laser. Nature communications, 6.
slide-57
SLIDE 57

What is 30fs?

100 fs  second Second  1000000 years Light travels 9 μm in 30fs

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-58
SLIDE 58

Experimental results

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-59
SLIDE 59

Protein quake

6/24/2016 Time resolved scattering studies - C. Blanchet

slide-60
SLIDE 60

Conclusion

  • SAXS is a good tool for time resolved

experiments

  • Good control on the initiation of the reaction

needed

  • Use experimental setup adapted to your

system

– Reaction triggering – time scale

6/24/2016 Time resolved scattering studies - C. Blanchet