Biology: Time-Resolved Solution Scattering

Time-resolved solution scattering is a very 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 solution scattering at BioCARS has been driven by user interest and needs, following the first successful wide-angle solution scattering (WAXS) experiments with ns time resolution conducted at the ID09 beamline, ESRF (Cammarata et al., 2008). BioCARS staff, in collaboration with Philip Anfinrud (NIH/NIDDK), implemented the infrastructure for time-resolved solution scattering experiments at 14ID beamline. 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). In addition to the improved time resolution, the q range was also extended as compared to the early ESRF studies to cover the broad range of 0.03-2.5Å-1, therefore providing simultaneous coverage of both SAXS and WAXS regions. These initial 100ps time-resolved studies at 14ID were conducted with myoglobin and dimeric hemoglobin samples. They compared and contrasted structural response to ligand photo-dissociation in solution to results of similar earlier time-resolved crystallographic studies (Srajer et al, 1996; Srajer et al., 2001, Schotte et al., 2003; Knapp et al., 2006; Knapp et al., 2009; Ren et al., 2012).

In the spring of 2014 the old MAR165 detector was replaced by a much larger-area and faster-readout Rayonix MX 340 HS detector. The new detector provides possibility for expanding the q-range for simultaneous SAXS/WAXS and significantly speeds up SAXS/WAXS data collection.

For time-resolved SAXS/WAXS experiments at BioCARS, users typically use a sealed capillary that can be translated rapidly during the experiment to refresh the sample exposed to the X-ray beam. A flow cell is also available. Reaction is typically triggered by short (ns or ps) laser pulses. BioCARS staff is currently developing infrastructure for rapid mixing approaches for reaction initiation.

 


 

Science Highlight

Heikki Takala, Alexander Björling, Oskar Berntsson, Heli Lehtivuori, Stephan Niebling, Maria Hoernke, Irina Kosheleva, Robert Henning, Andreas Menzel, Janne A. Ihalainen, and Sebastian Westenhoff, "Signal amplification and transduction in phytochrome photosensors," Nature 509, 245 (8 May 2014). DOI:10.1038/nature13310

Sensory proteins must relay structural signals from the sensory site over large distances to regulatory output domains. Phytochromes are a major family of red-light-sensing kinases that control diverse cellular functions in plants, bacteria and fungi. Bacterial phytochromes consist of a photosensory core and a carboxy-terminal regulatory domain. Structures of photosensory cores are reported in the resting state and conformational responses to light activation have been proposed in the vicinity of the chromophore. However, the structure of the signalling state and the mechanism of downstream signal relay through the photosensory core remain elusive. Here we report crystal and solution structures of the resting and activated states of the photosensory core of the bacteriophytochrome from Deinococcus radiodurans. The structures show an open and closed form of the dimeric protein for the activated and resting states, respectively. This nanometre-scale rearrangement is controlled by refolding of an evolutionarily conserved ‘tongue’, which is in contact with the chromophore. The findings reveal an unusual mechanism in which atomic-scale conformational changes around the chromophore are first amplified into an ångstrom-scale distance change in the tongue, and further grow into a nanometre-scale conformational signal. The structural mechanism is a blueprint for understanding how phytochromes connect to the cellular signalling network.

 

Lights, Conformational Change… Action!

 

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