Periodic Reporting for period 4 - LIFETimeS (Light-Induced Function: from Excitation to Signal through Time and Space)
Reporting period: 2023-03-01 to 2024-02-29
Organisms of all domains of life are capable of sensing, using and responding to light. This is achieved through specific proteins which contain a molecule or a molecular aggregate (the pigment(s)) able to absorb light in selected regions of the visible spectrum. The molecular mechanisms used are diverse, but the starting event is an electronic excitation localized on the pigment(s). This initial excitation rapidly “travels” across space to be converted in other forms of energy to complete the biological function.
In this project, we propose to create a single theoretical and computational framework able to “follow” the action of light from the initial electronic excitation up to the final biological function.
Theoretically addressing this cascade of processes calls for new models and computational strategies able to reproduce the dynamics across multiple space and time scales. Such a goal is formidably challenging as the interactions and the dynamics involved at each scale follow completely different laws, from those of the quantum world to those of classical particles.
Through the novel point of observation that will be achieved in the project, it will be possible not only to reveal the ‘design principles’ used by Nature but also to give a “practical” instrument to test “in silico” new techniques for the control of cellular processes by manipulating protein functions through light.
In this project, we propose to create a single theoretical and computational framework able to “follow” the action of light from the initial electronic excitation up to the final biological function.
Theoretically addressing this cascade of processes calls for new models and computational strategies able to reproduce the dynamics across multiple space and time scales. Such a goal is formidably challenging as the interactions and the dynamics involved at each scale follow completely different laws, from those of the quantum world to those of classical particles.
Through the novel point of observation that will be achieved in the project, it will be possible not only to reveal the ‘design principles’ used by Nature but also to give a “practical” instrument to test “in silico” new techniques for the control of cellular processes by manipulating protein functions through light.
The research has been developed along two distinct but connected directions. The first is dedicated to the advancement of new computational methodologies and algorithms, while the second focuses on the study of photosensitive proteins. We developed a novel multiscale strategy which combines quantum chemical and classical models and integrate the resulting hybrid approach into molecular dynamics simulations. This computational machinery has been applied to four different photoresponsive biological systems.
1) Phytochromes are ubiquitous photoreceptors responsible for sensing light in plants, fungi and bacteria. It is known that their photoactivation is initiated by the photoisomerization of the embedded bilin chromophore, which triggers a large conformational change in the protein but, although the numerous studies present in the literature, the molecular details of the whole photoactivation process remain elusive. By applying the newly developed computational machinery we have revealed that the chromophore photoisomerization proceeds through a mechanism whose kinetics is mainly determined by the network of hydrogen bonds in the binding pocket. The resulting photoproduct rapidly relaxes in an early intermediate, and finally evolves into a late intermediate, characterized by a more disordered binding pocket and a weakening of the salt-bridge interaction, whose cleavage is essential to interconvert the phytochrome to the final active state.
2) Blue Light-Using Flavin (BLUF) proteins are among the most important examples of blue-light-sensing flavoproteins. Among the BLUF systems, we have focused on Appa because there is still a large debate in the literature not only about its photoactivation but also on the real structure of the dark state. Previous studies strongly suggest that a Proton-Coupled Electron Transfer (PCET) process occurs at the excited state for several BLUF domains. However, this PCET process has never been experimentally proven for AppA. By using the newly developed computational machinery we have shown that the PCET mechanism is indeed possible for the AppA protein, suggesting a conserved mechanism among different BLUF domains.
3) In cyanobacteria, the light-harvesting of large antenna complexes called phycobilisomes (PBS) is accompanied by photoprotective mechanisms which are activated by strong blue-green light. A major role is played by the orange carotenoid protein (OCP) which contains a ketocarotenoid as light-sensitive component. Its photoactivation involves structural changes which terminate with an opening of OCP into two separate domains, one of which acts as a quencher for the PBS. Many experimental studies have tried to reveal the OCP’s action, but many fundamental aspects remain unclear. By using the newly developed computational machinery we have revealed the photoactivation mechanism in which the carotenoid does not act as a spring that, releasing its internal strain, induces the dissociation, as was previously proposed, but as a “latch” locking together the two domains. Finally, we have revealed how OCP binds to PBS and dissipates excess energy as heat in intense light conditions.
4) In plants, light-harvesting complexes serve as antennas to collect and transfer the absorbed energy to reaction centers, but also regulate energy transport by dissipating the excitation energy of chlorophylls. This process, known as nonphotochemical quenching, seems to be activated by conformational changes within the light-harvesting complex, but the quenching mechanisms remain elusive. By applying the newly developed computational machinery we have demonstrated that the protein scaffold acts as a molecular switch to activate or deactivate the quenching mechanism through conformational changes that induces distortions in the geometries of the embedded pigments and modifications on the electric fields acting on them.
1) Phytochromes are ubiquitous photoreceptors responsible for sensing light in plants, fungi and bacteria. It is known that their photoactivation is initiated by the photoisomerization of the embedded bilin chromophore, which triggers a large conformational change in the protein but, although the numerous studies present in the literature, the molecular details of the whole photoactivation process remain elusive. By applying the newly developed computational machinery we have revealed that the chromophore photoisomerization proceeds through a mechanism whose kinetics is mainly determined by the network of hydrogen bonds in the binding pocket. The resulting photoproduct rapidly relaxes in an early intermediate, and finally evolves into a late intermediate, characterized by a more disordered binding pocket and a weakening of the salt-bridge interaction, whose cleavage is essential to interconvert the phytochrome to the final active state.
2) Blue Light-Using Flavin (BLUF) proteins are among the most important examples of blue-light-sensing flavoproteins. Among the BLUF systems, we have focused on Appa because there is still a large debate in the literature not only about its photoactivation but also on the real structure of the dark state. Previous studies strongly suggest that a Proton-Coupled Electron Transfer (PCET) process occurs at the excited state for several BLUF domains. However, this PCET process has never been experimentally proven for AppA. By using the newly developed computational machinery we have shown that the PCET mechanism is indeed possible for the AppA protein, suggesting a conserved mechanism among different BLUF domains.
3) In cyanobacteria, the light-harvesting of large antenna complexes called phycobilisomes (PBS) is accompanied by photoprotective mechanisms which are activated by strong blue-green light. A major role is played by the orange carotenoid protein (OCP) which contains a ketocarotenoid as light-sensitive component. Its photoactivation involves structural changes which terminate with an opening of OCP into two separate domains, one of which acts as a quencher for the PBS. Many experimental studies have tried to reveal the OCP’s action, but many fundamental aspects remain unclear. By using the newly developed computational machinery we have revealed the photoactivation mechanism in which the carotenoid does not act as a spring that, releasing its internal strain, induces the dissociation, as was previously proposed, but as a “latch” locking together the two domains. Finally, we have revealed how OCP binds to PBS and dissipates excess energy as heat in intense light conditions.
4) In plants, light-harvesting complexes serve as antennas to collect and transfer the absorbed energy to reaction centers, but also regulate energy transport by dissipating the excitation energy of chlorophylls. This process, known as nonphotochemical quenching, seems to be activated by conformational changes within the light-harvesting complex, but the quenching mechanisms remain elusive. By applying the newly developed computational machinery we have demonstrated that the protein scaffold acts as a molecular switch to activate or deactivate the quenching mechanism through conformational changes that induces distortions in the geometries of the embedded pigments and modifications on the electric fields acting on them.
When considering the many different photosensing proteins present in living organisms, an image of evolutionary flexibility emerges, with examples of fusion proteins from multiple types of photosensors and photosensitive domains shared among diverse arrays of proteins. However, large questions remain: what are the ‘design principles’ used by Nature? Are there common themes, or is each solution truly unique?
Addressing these queries is significant not only for enhancing our comprehension of the molecular mechanisms underlying biological photoresponses, but also for the potential to modulate the activity of biosystems in vivo. This includes the capability to noninvasively manipulate protein functions with an unparalleled degree of spatiotemporal precision through the use of light.
In the project, a novel theoretical framework has been developed and transformed into new computational algorithms and new tools for the analyses of the simulations.
Thanks to this new theoretical and computational framework, it has been possible to “follow” the action of light, from the initial ultrafast electronic process localized at the subnanoscale, up to the final biological function covering time scales of the order of milliseconds.
The completeness and accuracy reached by the simulations based on such a new machinery has represented a breakthrough in our understanding of the mechanisms which govern the light-driven bioactivity.
Addressing these queries is significant not only for enhancing our comprehension of the molecular mechanisms underlying biological photoresponses, but also for the potential to modulate the activity of biosystems in vivo. This includes the capability to noninvasively manipulate protein functions with an unparalleled degree of spatiotemporal precision through the use of light.
In the project, a novel theoretical framework has been developed and transformed into new computational algorithms and new tools for the analyses of the simulations.
Thanks to this new theoretical and computational framework, it has been possible to “follow” the action of light, from the initial ultrafast electronic process localized at the subnanoscale, up to the final biological function covering time scales of the order of milliseconds.
The completeness and accuracy reached by the simulations based on such a new machinery has represented a breakthrough in our understanding of the mechanisms which govern the light-driven bioactivity.