Biology: Time-Resolved Solution Scattering

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.

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: 7-15 keV, 12 keV standard
• polychromatic beam, ~ 300 eV bandwidth used for solution scattering

Q-range

• 0.015 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. 40,191–285 (2007)
• Koch,et al, Small-angle scattering: a view on the properties, structures and structural changes of biological macromolecules in solution. Q. Rev. Biophys. 36,147–227, (2003)
• Feigin, D. Svergun , Structure Analysis by Small-Angle X-Ray and Neutron Scattering. New York: Plenum . 1987