Biology: Time-Resolved Solution Scattering

Sample environment for Time-Resolved X-Rays Solution Scattering setup at BioCARS.

Time-resolved solution scattering is an important component of the overall efforts at BioCARS to address dynamic aspects of macromolecular function. One of the most significant advantages of X-ray solution scattering is the ability to study biological macromolecules under near-physiological conditions (pH or ionic strength, for example) in the absence of crystal packing constraints. Development of time-resolved X-ray solution scattering (TRXSS) at BioCARS has been driven by user interest and need. BioCARS staff, in collaboration with Philip Anfinrud (NIH/NIDDK), implemented the infrastructure for time-resolved solution scattering experiments at 14ID beamline. BioCARS TRXSS setup allows simultaneous collection of SAXS/WAXS difference signal. The effort resulted in first solution scattering studies with 100ps time resolution (Cho et al., 2010; Kim J et al., 2011; Kim KH et al., 2011; Kim et al., 2012). These initial TRXSS experiments focused on myoglobin  and  dimeric hemoglobin molecules and were followed by studies of signaling pathways in photoactive yellow protein ( Cho at al., 2016) and bacteriophytochrome (Takkala et al., 2014, Björling et al., 2016). Most recent experiments utilized T-jumps induced by the infrared ns laser pulses and pH-jumps by using photo-caged protons. First T-jump TRXSS experiments studied details of insulin association and dissociation dynamics (Rimmerman et al., 2017, Rimmerman et al., 2018a). Using photo-acids for inducing pH–jump  resulted in unprecedented nanosecond time-resolution in SAXS protein folding-unfolding dynamic studies (Rimmerman et al., 2018b), not available yet in rapid-mixing experiments.

Scientific Contacts

Irina Kosheleva
Beamline Scientist
(630) 252-0467
ikoshelev@cars.uchicago.edu

The effort resulted in first solution scattering studies with 100ps time resolution (Cho et al., 2010; Kim J et al., 2011; Kim KH et al., 2011; Kim et al., 2012). These initial TRXSS experiments focused on myoglobin  and  dimeric hemoglobin molecules and were followed by studies of signaling pathways in photoactive yellow protein ( Cho at al., 2016) and bacteriophytochrome (Takkala et al., 2014, Björling et al., 2016). Most recent experiments utilized T-jumps induced by the infrared ns laser pulses and pH-jumps by using photo-caged protons. First T-jump TRXSS experiments studied details of insulin association and dissociation dynamics (Rimmerman et al., 2017, Rimmerman et al., 2018a). Using photo-acids for inducing pH–jump  resulted in unprecedented nanosecond time-resolution in SAXS protein folding-unfolding dynamic studies (Rimmerman et al., 2018b), not available yet in rapid-mixing experiments.

Technical capabilities

  • Time resolution
    • 100ps resolution in hybrid and 24-bunch APS storage ring mode
    • 200ns in 324-bunch APS mode
  • X-ray source
    • two in-line undulators (U23 and U27), optimized for 12 keV,
    • tunable: 7-16 keV, 12 keV standard
    • polychromatic beam, ~ 300 eV bandwidth used for solution scattering
  • Q-range
    • 015 to 4 Å-1 at 12 keV
    • suitable for molecules ~150Å in size, radius of gyration up to 57 Å (globular proteins)
    • camera lengths 180mm and 360mm
  • Sample delivery
    • quartz capillary, 10-µm wall thickness ( for reversible reactions)
    • quartz capillary flow cell, 10-µm wall thickness, connected by FTE or PEEK tubing to a syringe ( irreversible or slow reactions).
    • capillary diameter: 300-700 µm
    • thermal stabilization cell developed in collaboration with Lin Chen group ( Northwestern University) is available on request; temperature range: 10 -70°C
  • Lasers for reaction initiation:
    • ps laser system: Spectra Physics, Ti:Sapphire Spitfire Pro 5; 780nm; 2ps; 1kHz; 5mJ/pulse; TOPAS OPA; tunable range: 350nm-2µm; pulses typically stretched to 30ps
    • ns laser: OPOTEK Opolette 355 II HE; 7ns pulse duration; 20Hz; 410-600nm: ~3-6mJ; 230-400nm:0.5-2mJ
    • a number of CW diode lasers; ms exposures possible
  • Detector
    • Rayonix MX340-HS (10-100 frames/sec)

Conducting time-resolved experiments at BioCARS

TRXSS experiments are difference measurements. The reaction in the sample is initiated by short 30 ps or 7 ns  laser pulse at suitable wavelength. After specified time-delay, an X-ray pulse of 100 ps to 3.6 µs duration is used as a probe. Sample is then refreshed by flowing or by translating the capillary to the adjacent fresh spot. The pump-probe process is repeated until desired signal-to-noise ratio is achieved and detector is read out. A no-laser or negative time delay (where X-ray pulse precedes the laser pulse) image is also collected. Such reference image is subtracted from the image at each (positive) time delay to obtain difference time-resolved signal. On average for irreversible reaction in 300 µm diameter capillary ~30 µL of sample solution is required to obtain one image with 100 ps time resolution and ~ 1µL to obtain an image with ~4 µs time resolution. Depending on the sample concentration and the amplitude of the time-resolved signal, tens to hundreds images are necessary to collect to obtain high signal-to-noise ratio required for good quality data, particularly in the WAXS q-range.

Experimental data are reduced to 1D SAXS curve by in-house software on-the-fly. The resulting difference data can also be analyzed by the singular value decomposition and can be fit globally to determine kinetic constants.

Examples of structural analysis of BioCARS TRXSS data

  • Kim et al, Direct Observation of Cooperative Protein Structural Dynamics of Homodimeric Hemoglobin from 100 ps to 10 ms with Pump–Probe X-ray Solution Scattering, J. Am. Chem. Soc.134 (16), 7001-7008 (2012); also in Supplemental material
  • Bjorning et al, Deciphering Solution Scattering Data With Experimentally Guided Molecular Dynamic Simulations, JCTC, 11, 780-787 (2015); tutorial is at http://www.csb.gu.se/~alexander/gMD/
  • S. Cho et al, Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-Resolved Small- and Wide-Angle X-ray Scattering.
    Journal of the American Chemical Society 138, 8815–8823 (2016)

General information about SAXS technique

  • Blanchett and D.Svergun, Small Angle X-Ray Scattering on Biological Macromolecules and Nanocomposites in Solution. Ann. Rev. Phys. Chem. 64, 37-54 (2013)
  • Putnam et al,.X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q. Rev. Biophys. 40,191285 (2007)
  • Koch,et al, Small-angle scattering: a view on the properties, structures and structural changes of biological macromolecules in solution. Q. Rev. Biophys. 36,147227, (2003)
  • Feigin, D. Svergun , Structure Analysis by Small-Angle X-Ray and Neutron Scattering. New York: Plenum . 1987

T-Jump and PH jump experiment at BioCARS

D. Rimmermann et al, J. Phys. Chem. Lett. 8, 4413–4418 (2017), Rimmermann et al, J. Phys. Chem. B 122, 5218–5224 (2018)

Insulin hexamer dissociation dynamics revealed by photoinduced T-jumps and time-resolved X-ray solution scattering. a) Representative Kratky curves for bovine insulin under low pH conditions in aqueous-EtOH solution at 15, 30 and 50 °C representing the three major species. (b) SVD decomposition results from Kratky plot analysis.

D. Rimmerman, Photochemical & Photobiological Sciences, 7, 874-882, (2018)

Quaternary Structure Transitions in R-State Carbonmonoxyhemoglobin Unveiled in Time-Resolved X-ray Scattering Patterns Following a Temperature Jump

H.S. Cho et al, J.Phys.Chem.B, 122(49), 11488-11496,(2018)