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 understand how proteins work • Also had limits: • Same functions are performed by protein with different structures • IDP: some protein without structure still have functions What about looking directly at proteins in action?
Time resolved experiment Study systems whose structures change over time
Time scale of biological processes (protein folding) • Different time scale • Necessitates different kind of experiments
Generalities
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
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…
Pressure jump Woenckhaus, J., Köhling, R., Thiyagarajan, P., Littrell, K. C., Seifert, S., Royer, C. A., & Winter, R. (2001). Illustration: Dave, K., & Gruebele, M. (2015). Fast-folding proteins under stress. Cellular and Pressure-jump small-angle x-ray scattering detected kinetics of staphylococcal nuclease folding. Molecular Life Sciences , 72 (22), 4273-4285. Biophysical journal , 80 (3), 1518-1523.
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.
Light triggering Indirectly by releasing caged compounds Light acting directly on the protein Piant, S., Bolze, F., & Specht, A. (2016). Two-photon uncaging, from neuroscience to materials. Optical Materials Express , 6 (5), 1679-1691.
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 1.2 1.2 1.2 1 1 1 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 0 50 100 150 200 0 0 50 50 100 100 150 150 200 200
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) * Small measurement cell helps. 10/28/2019 Time resolved scattering studies - C. Blanchet
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
Continuous vs pump-probe Continuous Perturbation Probe ∆ t t Perturbation Probe Pump-probe ∆ t t 10/28/2019 Time resolved scattering studies - C. Blanchet
Continuous vs pump-probe ∆ t Perturbation Probe t Perturbation Probe ∆ t t 10/28/2019 Time resolved scattering studies - C. Blanchet
Continuous vs pump-probe ∆ t Perturbation Probe t Perturbation Probe ∆ t t 10/28/2019 Time resolved scattering studies - C. Blanchet
Limitation – Collection time 1 1 1 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 0 0 0 10 20 30 40 50 50 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 100 100 150 150 200 200 10/28/2019 Time resolved scattering studies - C. Blanchet
Limitation – Collection time 10/28/2019 Time resolved scattering studies - C. Blanchet
Short collection time: 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 • Pink beam 0 0 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 10/28/2019 Time resolved scattering studies - C. Blanchet Multilayer monochromator
DMM beam of P12 For protein: BSA 2.5 mg/ml 1.35 ms exposure time 100us exposure time
chopper P12 Chopper 2 modes: Stroboscopic pump and probe Production of short intense beam pulse Control of sample exposure (to limit radiation damage) Improve time resolution Detector collection X-ray pulse
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 60 ns • Gas detector (Theoretically, up to 1MHz) 10/28/2019 Time resolved scattering studies - C. Blanchet
Dead time of the reaction • Time between the reaction is triggered and the first point is collected (depends on triggering methods and collection time) 1 0.8 Short dead time required to study fast kinetic 0.6 0.4 0.2 0 10/28/2019 Time resolved scattering studies - C. Blanchet 0 50 100 150 200
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
Examples
Slow kinetics – Fibril formation 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 10/28/2019 Time resolved scattering studies - C. Blanchet
Amyloid fibrils • Insoluble protein aggregates • Implied in different diseases (Alzheimer, Parkinson, Type II diabetes,…) • Common structural features (cross beta) • Nucleation growth 10/28/2019 Time resolved scattering studies - C. Blanchet
SAXS Data • Singular value decomposition: 3 species 10/28/2019 Time resolved scattering studies - C. Blanchet
Models 10/28/2019 Time resolved scattering studies - C. Blanchet
Models 10/28/2019 Time resolved scattering studies - C. Blanchet
Sub-second kinetics • Stopped-flow (dead time: 2-10 ms) 10/28/2019 Time resolved scattering studies - C. Blanchet
MsbA nucleotide binding domain MsbA is an ATP-binding cassette Reaction of MsbA NBD with ATP transporter. that transports lipid A followed by SAXS and lipopolysaccharide through the inner membrane of Gram- Henning Tidow negative bacteria Inokentijs Josts Nucleotide binding domain (NBD) Illustration from Mi et al. Nature (2017) Josts et al. Structure (in press) 549: 233-237
Stopped flow triggering
MsbA nucleotide binding domain and ATP • Rapid mixing using stopped-flow • 35 ms frames collected with different delays after mixing
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.
ATP-induced transient dimerization of MsbA nucleotide binding domain Fit with a mixture of monomer and dimers
Calmodulin A Compact Intermediate State of Calmodulin in the Process of Target Binding. Yamada et al. Biochemistry 2012 Mastoparan 10/28/2019 Time resolved scattering studies - C. Blanchet
Caged compound release by flash photolysis • DM-nitrophen 10/28/2019 Time resolved scattering studies - C. Blanchet
Equilibrium measurement 10/28/2019 Time resolved scattering studies - C. Blanchet
Kinetics 10/28/2019 Time resolved scattering studies - C. Blanchet
140 ms 10 ms 0.5 ms 30 s With mastoparan Without mastoparan 10/28/2019 Time resolved scattering studies - C. Blanchet
Model 10/28/2019 Time resolved scattering studies - C. Blanchet
Ultra-fast time resolved 10/28/2019 Time resolved scattering studies - C. Blanchet
Ultra short collection time • Beamline ID09B, ESRF, Grenoble • Using the pulsed structure of the synchrotron • About 5000000 bunch/sec 10/28/2019 Time resolved scattering studies - C. Blanchet
Isolate one bunch • Isolate one bunch (ms shutter + fast chopper) 10/28/2019 Time resolved scattering studies - C. Blanchet
Single bunch experiment • High flux needed • Repetition of the measurements 10/28/2019 Time resolved scattering studies - C. Blanchet
Pump and probe experiment Trigger with Probe with Laser pulse X-ray τ t Bunch length ≈ 100 ps Resolution: up to 100 ps 10/28/2019 Time resolved scattering studies - C. Blanchet
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