Periodic Reporting for period 4 - MolStrucDyn (Ultrafast Molecular Structural Dynamics)
Okres sprawozdawczy: 2021-12-01 do 2023-05-31
The ERC project had the goal to visualize the femto- to nanosecond photoresponse of an important photosensor protein with time-resolved crystallography. The protein is called phytochrome and detects light in plants and various bacteria, thus providing the organisms with a sense to light. The project also aimed at uncovering new reaction pathways in chemistry with femtosecond diffusive X-ray scattering. Together, the project was designed to deliver "molecular movies" of chemical processes with ultrafast time and atomic structural resolution.
The methods used are state-of-the-art time-resolved X-ray scattering and crystallography. They open for detecting structural changes on ultrashort time-scales at which atoms move and perform chemical reactions.
The project has elucidated how photochemical reactions in solvents and in proteins are controlled by their respective molecular environment. Measuring molecular movies of chemical reactions at the femtosecond time scales is important for understanding chemical reactivity and thus the new knowledge has contributed to a better understanding of chemistry. In the longer term this 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.
The methods used are state-of-the-art time-resolved X-ray scattering and crystallography. They open for detecting structural changes on ultrashort time-scales at which atoms move and perform chemical reactions.
The project has elucidated how photochemical reactions in solvents and in proteins are controlled by their respective molecular environment. Measuring molecular movies of chemical reactions at the femtosecond time scales is important for understanding chemical reactivity and thus the new knowledge has contributed to a better understanding of chemistry. In the longer term this 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.
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.
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.
The project has made advancements beyond the state-of-the-art in three key areas, namely methodological improvements, insights into solution-state chemistry, and a deeper understanding of phytochromes photosensor proteins.
Methodological Advancements: The project has pushed the boundaries of time-resolved X-ray solution scattering, enabling the observation of photoactive molecules and chemical reactions with unprecedented structural detail. The project has not only resulted in new approaches for interpreting data in terms of molecular structure but also pioneered the integration of theoretical modelling and simulations with experimental data.
Real-Time Bond Breaking: Utilizing these methods, the project achieved a ground-breaking milestone by providing the first real-time and real-space observations of bond breaking in a solution. The data allowed us to track the progressive elongation and eventual rupture of chemical bonds, along with the subsequent reorganization of separated atoms. This entire process was found to be intricately mediated by the solvent cage, defined by the surrounding solvent molecules.
Insights into Phytochromes: In the realm of phytochromes, which are photosensor proteins in plants, fungi, and bacteria, the project delivered the first-ever structural data on a phytochrome in its photoexcited state. Notably, we discovered that the protein environment's influence on phytochromes was exceptionally significant compared to other proteins, prompting fascinating questions regarding the reasons behind this disparity. These findings shed light on how proteins sense light and play crucial roles in governing the growth, photosynthesis, and behavior of plants and bacteria.
In summary, our research has provided novel experimental insights with structural specificity into how the surrounding environment, whether composed of solvent molecules or the protein matrix, exerts a profound impact on photoreactions. This knowledge enhances our understanding of how solvent molecules influence the outcomes of chemical reactions and underscores the finely tuned protein frameworks that nature has evolved to orchestrate essential enzymatic reactions vital for life.
Methodological Advancements: The project has pushed the boundaries of time-resolved X-ray solution scattering, enabling the observation of photoactive molecules and chemical reactions with unprecedented structural detail. The project has not only resulted in new approaches for interpreting data in terms of molecular structure but also pioneered the integration of theoretical modelling and simulations with experimental data.
Real-Time Bond Breaking: Utilizing these methods, the project achieved a ground-breaking milestone by providing the first real-time and real-space observations of bond breaking in a solution. The data allowed us to track the progressive elongation and eventual rupture of chemical bonds, along with the subsequent reorganization of separated atoms. This entire process was found to be intricately mediated by the solvent cage, defined by the surrounding solvent molecules.
Insights into Phytochromes: In the realm of phytochromes, which are photosensor proteins in plants, fungi, and bacteria, the project delivered the first-ever structural data on a phytochrome in its photoexcited state. Notably, we discovered that the protein environment's influence on phytochromes was exceptionally significant compared to other proteins, prompting fascinating questions regarding the reasons behind this disparity. These findings shed light on how proteins sense light and play crucial roles in governing the growth, photosynthesis, and behavior of plants and bacteria.
In summary, our research has provided novel experimental insights with structural specificity into how the surrounding environment, whether composed of solvent molecules or the protein matrix, exerts a profound impact on photoreactions. This knowledge enhances our understanding of how solvent molecules influence the outcomes of chemical reactions and underscores the finely tuned protein frameworks that nature has evolved to orchestrate essential enzymatic reactions vital for life.