0000000000437603
AUTHOR
Michael Wulff
Structural photoactivation of a full-length bacterial phytochrome
Time-resolved x-ray solution scattering reveals the conformational signaling mechanism of a bacterial phytochrome.
Unveiling the timescale of the R-T transition in human hemoglobin.
Time-resolved wide-angle X-ray scattering, a recently developed technique allowing to probe global structural changes of proteins in solution, was used to investigate the kinetics of R-T quaternary transition in human hemoglobin and to systematically compare it to that obtained with time-resolved optical spectroscopy under nearly identical experimental conditions. Our data reveal that the main structural rearrangement associated with the R-T transition takes place approximately 2 mus after the photolysis of hemoglobin at room temperature and neutral pH. This finding suggests that the 20-mus step observed with time-resolved optical spectroscopy corresponds to a small and localized structural…
Impulsive solvent heating probed by picosecond x-ray diffraction
The time-resolved diffraction signal from a laser-excited solution has three principal components: the solute-only term, the solute-solvent cross term, and the solvent-only term. The last term is very sensitive to the thermodynamic state of the bulk solvent, which may change during a chemical reaction due to energy transfer from light-absorbing solute molecules to the surrounding solvent molecules and the following relaxation to equilibrium with the environment around the scattering volume. The volume expansion coefficient alpha for a liquid is typically approximately 1 x 10(-3) K(-1), which is about 1000 times greater than for a solid. Hence solvent scattering is a very sensitive on-line t…
Light-induced structural changes in a monomeric bacteriophytochrome
International audience; Phytochromes sense red light in plants and various microorganism. Light absorption causes structural changes within the protein, which alter its biochemical activity. Bacterial phytochromes are dimeric proteins, but the functional relevance of this arrangement remains unclear. Here, we use time-resolved X-ray scattering to reveal the solution structural change of a monomeric variant of the photosensory core module of the phytochrome from Deinococcus radiodurans. The data reveal two motions, a bend and a twist of the PHY domain with respect to the chromophore-binding domains. Infrared spectroscopy shows the refolding of the PHY tongue. We conclude that a monomer of th…
Probing in cell protein structural changes with time-resolved X-ray scattering
International audience; Investigating protein structural changes inside the cell is a major goal in molecular biology. Here we show that time-resolved wide-angle X-ray scattering is a valuable tool for this purpose. Hemoglobin has been chosen as a model system and its tertiary and quaternary conformational changes following laser flash-photolysis have been tracked in intact red blood cells with nanosecond time resolution.
Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering
We demonstrate tracking of protein structural changes with time-resolved wide-angle X-ray scattering (TR-WAXS) with nanosecond time resolution. We investigated the tertiary and quaternary conformational changes of human hemoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. We also report data on optically induced tertiary relaxations of myoglobin and refolding of cytochrome c to illustrate the wide applicability of the technique. By providing insights into the structural dynamics of proteins functioning in their natural environment, TR-WAXS complements and extends results obtained with time-resolved optical spectroscopy and X-ray crystallography.
Spatiotemporal reaction kinetics of an ultrafast photoreaction pathway visualized by time-resolved liquid x-ray diffraction.
We have studied the reaction dynamics for HgI 2 in methanol by using time-resolved x-ray diffraction (TRXD). Although numerous time-resolved spectroscopic studies have provided ample information about the early dynamics of HgI 2 , a comprehensive reaction mechanism in the solution phase spanning from picoseconds up to microseconds has been lacking. Here we show that TRXD can provide this information directly and quantitatively. Picosecond optical pulses triggered the dissociation of HgI 2 , and 100-ps-long x-ray pulses from a synchrotron probed the evolving structures over a wide temporal range. To theoretically explain the diffracted intensities, the structural signal from the solute, the…
Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering
Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca2] E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release o…