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Ultrafast Molecular Structural Dynamics

Periodic Reporting for period 2 - MolStrucDyn (Ultrafast Molecular Structural Dynamics)

Reporting period: 2018-12-01 to 2020-05-31

"The ERC project has the goal to visualize the femto- to nanosecond photoresponse of an important photosensor protein with time-resolved crystallography. It also aims at uncovering new reaction pathways in chemistry with femtosecond diffusive X-ray scattering. The project is designed to deliver ""molecular movies"" of the chemical processes, which occur directly after triggering of the reactions.

The methods used are state-of-the-art time-resolved X-ray scattering and crystallography. They open up for detecting structural changes on femtosecond time-scales.

Measuring molecular movies of chemical reactions at the femtosecond time scales is important for understanding chemical reactivity. The new knowledge will contribute to a better understanding of chemistry, which can lead to novel materials, novel drugs, or more cost- or environmentally friendly production of chemicals. Understanding the initial photoresponse of phytochrome proteins is important, because the proteins steer the growth and development of all plants on earth. The optimal development of plants is of fundamental importance for all vegetation on earth. Together the project will elucidate how photochemical reactions in solvents and in proteins are controlled by their respective molecular environment."
We have performed a number of time-resolved X-ray scattering experiments at X-ray free electron lasers. In the epxeriment, a photoactive chemical in solution is photoexcited using a short laser pulse in the UV or visible spectral region. A short X-ray pulse than scatters of the sample at a defined time gap compared to the excitation pulse. From the time-dependent scattering, structural information about the chemicals can be obtained. The experiments are state-of-the-art and have a time-resolution of femtoseconds. The data reveal the structural changes of chemicals on time scales when chemical bonds are broken and formed. A major effort was put into the structural analysis of the data. This is currently the bottleneck for the technique and we believe that we have demonstrated novel concepts and implementation for data analysis, which make the technique more viable.

We have also performed time-resolved crystallographic studies of a phytochrome proteins, which are important photoreceptor proteins in plants, fungi, and bacteria. The proteins detects light in plants, providing the plant with crucial information about environmental conditions. With this information, the plants can guide growth and development. The time-resolved crystallographic experiment is very similar to the X-ray scattering experiment described above, except for the the protein is supplied as small micormeter-sized crystals. The data obtained are time-dependent crystallographic patterns, which report on structural changes in the protein. We have performed this experiment successfully for two phytochromes. The data reveal the first structural information on the excited state of the phytochrome. The changes are important to understand to be able to gasp how the proteins convert light signals into biochemical signals, which are passed on to the cell and the organism.

WP1 has been published as Woitowitch et al. IuPCr 2018, Sanchez et al. Structural Dynamics 2019, and Claesson et al., 2019, BioRxiv & submitted). The work has been presented at conference, for example: Light and LIfe, Barcelona, 2019, BioXFEL 5th conference, San Juan, 2020, and the ACA meeting 2019, Cincinnati, Westenhoff.
The project has pushed time-resolved X-ray solution scattering beyond its limits for the study of photoactive molecules. With this, we have followed chemical reactions with unprecedented structural details. We have demonstrated the first structural data on a phytochrome in its photoexcited state. this provides valuable understanding on how the proteins detect light and with that guide the growth, photosynthesis, and behavior of vegetation and bacteria.

Our first results indicate that we will identify the structural mechanism of signal activation for phytochrome proteins; that we will contribute new understanding for chemical reaction dynamics on 'chemical' femtosecond time scales, and that we will understand how the environment of the photoactive molecules, which could be solvent molecules or the proteins matrix, influences the photoreaction. This will provide much needed understanding for how nature has tuned protein scaffolds to direct enzymatic reactions for life.