Time resolved SAXS Clement Blanchet Foreword Structural biology: - - PowerPoint PPT Presentation

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Time resolved SAXS Clement Blanchet Foreword Structural biology: - - PowerPoint PPT Presentation

Mar 31, 2024 606 likes •1.18k views

Time resolved SAXS Clement Blanchet Foreword Structural biology: knowing the structure to understand the function Structure Function Works quite well, we now have a lot of protein structure that shed light on their functions and help to

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Time resolved SAXS

Clement Blanchet

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Foreword

  • Structural biology: knowing the structure to understand the function
  • Works quite well, we now have a lot of protein structure that shed

light on their functions and help to understand how proteins work

  • Also had limits:
  • Same functions are performed by protein with different structures
  • IDP: some protein without structure still have functions

Structure

Function What about looking directly at proteins in action?

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Time resolved experiment

Study systems whose structures change over time

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Time scale of biological processes

(protein folding)

  • Different time scale
  • Necessitates different kind of experiments
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Generalities

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Time resolved experiments

  • Reaction of interest needs to be properly controlled:
  • Controlled triggering of the reaction of interest.
  • System at equilibrium is perturbed, and one monitor the return to a new

equilibrium

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Triggering the reaction

Changing the chemical composition of your solvent (Mixing your solution with a reactant)

  • Change of pH, salt concentration, introducing ligand or interacting ions…
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Pressure jump

Illustration: Dave, K., & Gruebele, M. (2015). Fast-folding proteins under stress. Cellular and Molecular Life Sciences, 72(22), 4273-4285.

Woenckhaus, J., Köhling, R., Thiyagarajan, P., Littrell, K. C., Seifert, S., Royer, C. A., & Winter, R. (2001). Pressure-jump small-angle x-ray scattering detected kinetics of staphylococcal nuclease folding. Biophysical journal, 80(3), 1518-1523.

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Temperature jump

  • By mixing
  • Using laser pulse

Kubelka, J. (2009). Time-resolved methods in biophysics. 9. Laser temperature-jump methods for investigating biomolecular dynamics. Photochemical & Photobiological Sciences, 8(4), 499-512.

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Light triggering

Light acting directly on the protein Indirectly by releasing caged compounds

Piant, S., Bolze, F., & Specht, A. (2016). Two-photon uncaging, from neuroscience to materials. Optical Materials Express, 6(5), 1679-1691.

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How fast the reaction should be triggered depends on how fast the system reacts.

  • Triggering:
  • Simultaneous, fast and homogeneous triggering at the time

scale of the reaction

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

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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)

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* Small measurement cell helps.

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Monitor 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
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Continuous vs pump-probe

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t t

Perturbation Perturbation Probe Probe

∆t ∆t

Continuous Pump-probe

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Continuous vs pump-probe

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t t

Perturbation Perturbation Probe Probe

∆t ∆t

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Continuous vs pump-probe

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t t

Perturbation Perturbation Probe Probe

∆t ∆t

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Limitation – Collection time

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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

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Limitation – Collection time

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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

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Undulator Double crystal monochromator Multilayer monochromator

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DMM beam of P12

For protein: BSA 2.5 mg/ml 1.35 ms exposure time 100us exposure time

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chopper

  • Production of short intense beam pulse
  • Control of sample exposure (to limit

radiation damage)

  • Improve time resolution

Detector collection X-ray pulse

P12 Chopper

  • 2 modes:
  • Stroboscopic
  • pump and probe
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Short collection time - Fast detector

  • Photon counting detector: Pilatus (300Hz), Eiger (up to 2kHz)
  • Detector gating: condition when the detector can count photons on an

external signal

  • Gas detector (Theoretically, up to 1MHz)

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60 ns

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Dead time of the reaction

  • Time between the reaction is triggered and the first point is collected

(depends on triggering methods and collection time)

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0.2 0.4 0.6 0.8 1 50 100 150 200

Short dead time required to study fast kinetic

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Practical tips

  • Know your reaction
  • Master your triggering method
  • Know your time scale
  • A lot of sample is required
  • Depends on:
  • Radiation damage
  • Number of point in the kinetics
  • Signal to noise ratio desired
  • At least 1 ml of sample, often more, is required
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Examples

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Slow kinetics – Fibril formation

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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

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Amyloid fibrils

  • Insoluble protein aggregates
  • Implied in different diseases (Alzheimer, Parkinson, Type II

diabetes,…)

  • Common structural features (cross beta)
  • Nucleation growth

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SAXS Data

  • Singular value decomposition:

3 species

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Models

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Models

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Sub-second kinetics

  • Stopped-flow (dead time: 2-10 ms)

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MsbA nucleotide binding domain

Illustration from Mi et al. Nature (2017) 549: 233-237

MsbA is an ATP-binding cassette

  • transporter. that transports lipid A

and lipopolysaccharide through the inner membrane of Gram- negative bacteria

Nucleotide binding domain (NBD)

Henning Tidow Inokentijs Josts

Josts et al. Structure (in press)

Reaction of MsbA NBD with ATP followed by SAXS

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Stopped flow triggering

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MsbA nucleotide binding domain and ATP

  • Rapid mixing using stopped-flow
  • 35 ms frames collected with

different delays after mixing

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MsbA nucleotide binding domain and ATP

In the first phase (t<2.5s), rapid increase of the radius of gyration, then (t>2.5s) slow decrease.

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ATP-induced transient dimerization of MsbA nucleotide binding domain

Fit with a mixture of monomer and dimers

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Calmodulin

A Compact Intermediate State of Calmodulin in the Process of Target

  • Binding. Yamada et al. Biochemistry 2012

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Mastoparan

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Caged compound release by flash photolysis

  • DM-nitrophen

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Equilibrium measurement

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Kinetics

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

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Model

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Ultra-fast time resolved

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Ultra short collection time

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

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Isolate one bunch

  • Isolate one bunch (ms shutter + fast chopper)

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Single bunch experiment

  • High flux needed
  • Repetition of the measurements

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Pump and probe experiment

t

Trigger with Laser pulse Probe with X-ray τ

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

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TR WAXS

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

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T and R states of hemoglobin

Looking at the unbinding of oxygen by hemoglobin

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Experimental setup

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Structural change in hemoglobin

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FEL

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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.

Arnlund, D., Johansson, L. C., Wickstrand, C., Barty, A., Williams, G. J., Malmerberg, E., ... & Wang, D. (2014). Visualizing a protein quake with time-resolved X-ray scattering at a free- electron laser. Nature methods, 11(9), 923.

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What is 30fs?

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

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Experimental results

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Protein quake

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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

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