We have conducted a series of time-resolved X-ray scattering experiments at X-ray free-electron lasers. In these experiments, a photoactive chemical in solution is excited by a short laser pulse in the UV or visible spectral region. Subsequently, a short X-ray pulse scatters off the sample at a specific time delay after the excitation pulse. By analyzing the time-dependent scattering patterns, we obtained structural information about the chemicals with femtosecond time resolution. These experiments have provided insights into the structural changes of chemicals at the moment when chemical bonds are broken and formed. To overcome a major challenge in the method, we have devoted significant effort to developing a comprehensive data analysis pipeline, making the technique more accessible to other researchers.
In addition, we have conducted time-resolved crystallographic studies on phytochrome proteins, which are crucial photoreceptor proteins found in plants, fungi, and bacteria. These proteins play a vital role in light detection in plants, providing essential information about the environment for guiding growth and development. The experimental setup for time-resolved crystallography is similar to the X-ray scattering experiment described earlier, except that the protein is supplied in the form of small micrometer-sized crystals. The collected data consist of time-dependent crystallographic patterns that reveal structural changes in the protein. We have successfully performed this experiment on two different phytochromes, yielding the first structural insights into their excited states. Surprisingly, our results demonstrate that the protein's environment surrounding the photoexcited chromophore actively participates in the photoresponse on the femtosecond timescale. This suggests that evolutionary optimization extends beyond the amino acid sequence and 3D structure to include the dynamics of side chains and water molecules within the protein structure. Understanding these changes is crucial for unravelling how these proteins convert light signals into biochemical signals that are further transmitted to cells and organisms.
The findings related to photochemicals have been published in Panman et al., PRL 2021 and Nimmrich et al., JACS 2023. The research on phytochromes has been published in Sanchez et al., Structure 2021, Claesson et al., Elife 2020, with an upcoming publication. A review article on the subject was published as Westenhoff et al., Curr. Opp. Struc. Biol, 2022. Additionally, the work has been presented at six international conferences.
