Periodic Reporting for period 3 - LIFETimeS (Light-Induced Function: from Excitation to Signal through Time and Space)
Okres sprawozdawczy: 2021-09-01 do 2023-02-28
Organisms of all domains of life are capable of sensing, using and responding to light. This is achieved through the use of 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 and finally used to complete the biological function.
Here, 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 realized, 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.
Here, 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 realized, 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 the first reporting period we mostly focused on the formulation of an an hybrid which combines quantum chemical and classical models and its integration into molecular dynamics simulations. In the second and the third reporting periods this computational machinery has been applied to various systems:
1) The photosynthetic apparatus of higher plants can dissipate excess excitation energy during high light exposure, by deactivating excited chlorophylls through a mechanism called non- photochemical quenching. However, the precise molecular details are still not completely understood. We have shown that a charge transfer state involving a specific carotenoid pigment can efficiently quench chlorophyll excitation, and reduce the excitation lifetime of the major light-harvesting complex of Photosystem II. Through a combination of MD simulations, QM/MM calculations, and kinetic modeling, we have demonstrated that the quenching level can be finely tuned by the protein, by regulating the energy of the charge transfer state. Our results suggest that the protein scaffold can act as a molecular switch to activate or deactivate the quenching mechanism through small changes in its structural constraints and electric fields.
2) To adapt to different light conditions, photosynthetic cyanobacteria have developed photoregulatory mechanisms that allow them to modulate the amount of energy harvested and dissipate the excess in heat. These mechanisms are mediated by a protein, the so-called Orange Carotenoid Protein (OCP). Its photoactivation involves a series of structural changes which terminate with an opening of the complex into two separate domains, one of which acts as a quencher for the LH complexes. After having successfully reproduced the experimental UV-Vis and Raman spectra of the inactive and active forms of OCP, we have revealed the photoactivation mechanism by simulating at the atomistic level the whole dynamics of the complex through an effective combination of enhanced sampling techniques. On the basis of our findings, we can conclude that 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.
3) In plants, bacteria and fungi, red-light-sensing proteins (phytochromes) control diverse cellular functions. In these systems, the protein binds a tetrapyrrole chromophore (a bilin) which, absorbing light, acts as a switch between resting and active states. However, how the molecular mechanism si still poorly understood. Applying our computational machinery to a bacteriophytochrome, we have investigated both the inactive and the activated form, revealing a large structure relaxation in solution, compared to the crystal structures. By integrating classical MD and QM/MM nonadiabatic dynamics simulations we have also revealed that chromophore photoisomerization proceeds through a hula-twist mechanism whose kinetics is mainly determined by the network of hydrogen bonds in the pocket. The resulting photoproduct relaxes to an early intermediate stabilized, and finally evolves into a late intermediate, featuring a more disordered binding pocket.
4) A large class of photoreceptors uses flavin as chromophore. However, their mechanisms of photoactivation can be diverse. In Blue-Light Using Flavin (BLUF) domains both dark- and light-adapted structures present the same oxidized form of the flavin but with a change in the chemical environment of the chromophore. Among BLUF domains, a very special case is the AppA protein found in the bacterium Rhodobacter sphaeroides. Two crystallographic structures of AppA have been resolved, which differ in the residues forming the flavin binding pocket. To resolve this structural ambiguity, we have investigated the two proposed structures using MD simulations and NMR, IR, and UV-vis spectroscopic calculations. Our simulations confirmed that the dark-adapted state of AppA is compatible with the structure containing the methionine in the binding site instead of the tryptophan. Moving to the photoactivation mechanism, 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 polarizable QM/MM dynamics simulations in the excited and ground states we have shown that the PCET mechanism is indeed possible for the AppA protein, suggesting a conserved mechanism among different BLUF domains.
1) The photosynthetic apparatus of higher plants can dissipate excess excitation energy during high light exposure, by deactivating excited chlorophylls through a mechanism called non- photochemical quenching. However, the precise molecular details are still not completely understood. We have shown that a charge transfer state involving a specific carotenoid pigment can efficiently quench chlorophyll excitation, and reduce the excitation lifetime of the major light-harvesting complex of Photosystem II. Through a combination of MD simulations, QM/MM calculations, and kinetic modeling, we have demonstrated that the quenching level can be finely tuned by the protein, by regulating the energy of the charge transfer state. Our results suggest that the protein scaffold can act as a molecular switch to activate or deactivate the quenching mechanism through small changes in its structural constraints and electric fields.
2) To adapt to different light conditions, photosynthetic cyanobacteria have developed photoregulatory mechanisms that allow them to modulate the amount of energy harvested and dissipate the excess in heat. These mechanisms are mediated by a protein, the so-called Orange Carotenoid Protein (OCP). Its photoactivation involves a series of structural changes which terminate with an opening of the complex into two separate domains, one of which acts as a quencher for the LH complexes. After having successfully reproduced the experimental UV-Vis and Raman spectra of the inactive and active forms of OCP, we have revealed the photoactivation mechanism by simulating at the atomistic level the whole dynamics of the complex through an effective combination of enhanced sampling techniques. On the basis of our findings, we can conclude that 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.
3) In plants, bacteria and fungi, red-light-sensing proteins (phytochromes) control diverse cellular functions. In these systems, the protein binds a tetrapyrrole chromophore (a bilin) which, absorbing light, acts as a switch between resting and active states. However, how the molecular mechanism si still poorly understood. Applying our computational machinery to a bacteriophytochrome, we have investigated both the inactive and the activated form, revealing a large structure relaxation in solution, compared to the crystal structures. By integrating classical MD and QM/MM nonadiabatic dynamics simulations we have also revealed that chromophore photoisomerization proceeds through a hula-twist mechanism whose kinetics is mainly determined by the network of hydrogen bonds in the pocket. The resulting photoproduct relaxes to an early intermediate stabilized, and finally evolves into a late intermediate, featuring a more disordered binding pocket.
4) A large class of photoreceptors uses flavin as chromophore. However, their mechanisms of photoactivation can be diverse. In Blue-Light Using Flavin (BLUF) domains both dark- and light-adapted structures present the same oxidized form of the flavin but with a change in the chemical environment of the chromophore. Among BLUF domains, a very special case is the AppA protein found in the bacterium Rhodobacter sphaeroides. Two crystallographic structures of AppA have been resolved, which differ in the residues forming the flavin binding pocket. To resolve this structural ambiguity, we have investigated the two proposed structures using MD simulations and NMR, IR, and UV-vis spectroscopic calculations. Our simulations confirmed that the dark-adapted state of AppA is compatible with the structure containing the methionine in the binding site instead of the tryptophan. Moving to the photoactivation mechanism, 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 polarizable QM/MM dynamics simulations in the excited and ground states we have shown that the PCET mechanism is indeed possible for the AppA protein, suggesting a conserved mechanism among different BLUF domains.
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?
The importance of answering these questions lies not only in the possibility of achieving a better understanding of the molecular mechanisms at the basis of the biological photoresponse but also in that of controlling the activity of biosystems in vivo and being able to manipulate protein functions in a noninvasive way and with unprecedented spatiotemporal resolution using light.
Here, we propose to create a novel theoretical and computational framework that, for the first time, will be able 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. This description will be possible only if a new conceptual machinery is designed and transformed into new computational algorithms and new tools for the analyses of the simulations.
The completeness and accuracy reached by the simulations based on such a new machinery will represent a breakthrough in our understanding of the mechanisms which govern the light-driven bioactivity.
The importance of answering these questions lies not only in the possibility of achieving a better understanding of the molecular mechanisms at the basis of the biological photoresponse but also in that of controlling the activity of biosystems in vivo and being able to manipulate protein functions in a noninvasive way and with unprecedented spatiotemporal resolution using light.
Here, we propose to create a novel theoretical and computational framework that, for the first time, will be able 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. This description will be possible only if a new conceptual machinery is designed and transformed into new computational algorithms and new tools for the analyses of the simulations.
The completeness and accuracy reached by the simulations based on such a new machinery will represent a breakthrough in our understanding of the mechanisms which govern the light-driven bioactivity.