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.
