Periodic Reporting for period 2 - BioInspired_SolarH2 (Engineering Bio-Inspired Systems for the Conversion of Solar Energy to Hydrogen)
Período documentado: 2020-10-01 hasta 2022-03-31
The current energy approach is not sustainable. Our reliance on fossil fuels produces the dangerous accumulation of carbon dioxide in the atmosphere which has disastrous consequences for the environment and our health. Therefore, a change of paradigm is required. The solution resides in nature, more specifically in photosynthesis, the fundamental biological process by which solar energy, water and carbon dioxide are converted into oxygen and biomass.
Here, I propose to apply the detailed knowledge that we have gather over the years about the design principles that lead to efficient solar-energy collection, transfer and conversion in photosynthesis to engineer bio-inspired molecular machines able to convert solar energy to a separation of charges, that once coupled to catalysts, will drive water splitting and produce hydrogen, a carbon-neutral solar fuel.
The Design Principles of Photosynthetic Charge Separation are: i) collective excited states (excitons), ii) multiple charge-separation pathways, iii) coherent mixing between excitons and charge-transfer (CT) states promoted by resonant vibrations, iv) the smart protein matrix that controls the selection of the charge-separation pathways and the presence of coherence.
These Design Principles will be implemented into bio-inspired chromophore-protein assemblies that will be studied by a series of state-of-the-art spectroscopic techniques.
Therefore, the overall objective is to generate solar fuels with a system composed by renewable and abundant materials, utilizing water as starting material and solar energy as driving force.
Here, I propose to apply the detailed knowledge that we have gather over the years about the design principles that lead to efficient solar-energy collection, transfer and conversion in photosynthesis to engineer bio-inspired molecular machines able to convert solar energy to a separation of charges, that once coupled to catalysts, will drive water splitting and produce hydrogen, a carbon-neutral solar fuel.
The Design Principles of Photosynthetic Charge Separation are: i) collective excited states (excitons), ii) multiple charge-separation pathways, iii) coherent mixing between excitons and charge-transfer (CT) states promoted by resonant vibrations, iv) the smart protein matrix that controls the selection of the charge-separation pathways and the presence of coherence.
These Design Principles will be implemented into bio-inspired chromophore-protein assemblies that will be studied by a series of state-of-the-art spectroscopic techniques.
Therefore, the overall objective is to generate solar fuels with a system composed by renewable and abundant materials, utilizing water as starting material and solar energy as driving force.
Regarding Aim I: We have successfully constructed several chromophore-protein assemblies with different protein designs, based on a four-alpha-helix bundle structure. These are Alpha, AlphaMutant, AlphaTopDimer, AlphaBottomDimer, Beta, BetaMutant, BetaTopDimer, BetaBottomDimer, Omega, OmegaMutant, OmegaTopDimer, OmegaBottomDimer, and Epsilon. We have studied various properties of the chromophores and proteins independently: Thermal stability of the proteins, effect of salt concentration on the protein folding, and chromophore stability in organic solvent. Furthermore, we have investigated the properties of the assemblies: the chromophore-protein binding strength, the number of chromophores bound to the protein, and the interactions among the chromophores within the protein. These aspects have been investigated by a combination of spectroscopic techniques: Absorption, Fluorescence, Circular Dichroism, and Time-Correlated Single Photon Counting. At the moment we are working
on the implementation of two new techniques: Linear Dichroism and Stark spectroscopy.
Our results show that several protein designs can accommodate four chromophores (the maximum we expected) (i.e. Beta and Omega) and that these interact forming excitonically coupled dimers at the top and bottom of the structure in Beta and, most likely, at the top or bottom of the structure in Omega. Currently, we are investigating whether the excitonically coupled dimer in Omega is formed at the top or bottom of the structure. We have developed a novel protocol to efficiently create chromophore-protein assemblies with the maximum number of chromophores bound to the protein and complete excitonic interactions in the protein design Beta. To generate excitonically coupled dimers is especially exciting since it is the first step to generate functional chromophore-protein assemblies able to perform ultrafast and efficient energy and electron transfer, and consequently, it is the first step to convert solar energy to a separation of charges. Furthermore, we have investigated the thermal stability of the chromophore-protein assemblies (previously we investigated the thermal stability of the proteins “empty”, without the chromophores inserted) and, remarkably, we have observed a dramatic stabilization of the protein with the chromophores bound. For instance, the melting temperature (temperature at which half of the proteins within the sample ensemble are folded and half are unfolded) for Alpha increases from 37°C to 79°C, and for Beta from 50°C to 67°C, whereas for Epsilon (a control protein that cannot bind chromophores) the melting temperature does not change when chromophores are added to the solution, demonstrating that the thermal stabilization is promoted by the presence of the chromophores inside the protein. We are writing two manuscripts based on these results.
Aim 2 has not been started yet (it requires Aim 1 to be completed).
Regarding Aim 3: In parallel to Aims 1-2, this Aim intends to advance the understanding of the role of coherence in enhancing function in photosynthesis, that is, LH and CS in natural pigment-protein complexes. We have successfully performed Two-Dimensional Electronic Spectroscopy on two LH complexes isolated from plants: CP43 and CP47. We have obtained high-quality spectroscopic data which we are carefully processed and analyzed. The results allow us to unravel multiple energy transfer pathways within CP43/CP47 with unprecedented detail, never before the energy transfer from each excitonic band to the lowest energy states in these systems could have been resolved. Even more exciting, we have been able to demonstrate that energy transfer in CP43/CP47 proceeds via a vibration-assisted (vibronic) mechanism, a highly debated topic in the literature. Finally, we have written a scientific manuscript based on these results.
on the implementation of two new techniques: Linear Dichroism and Stark spectroscopy.
Our results show that several protein designs can accommodate four chromophores (the maximum we expected) (i.e. Beta and Omega) and that these interact forming excitonically coupled dimers at the top and bottom of the structure in Beta and, most likely, at the top or bottom of the structure in Omega. Currently, we are investigating whether the excitonically coupled dimer in Omega is formed at the top or bottom of the structure. We have developed a novel protocol to efficiently create chromophore-protein assemblies with the maximum number of chromophores bound to the protein and complete excitonic interactions in the protein design Beta. To generate excitonically coupled dimers is especially exciting since it is the first step to generate functional chromophore-protein assemblies able to perform ultrafast and efficient energy and electron transfer, and consequently, it is the first step to convert solar energy to a separation of charges. Furthermore, we have investigated the thermal stability of the chromophore-protein assemblies (previously we investigated the thermal stability of the proteins “empty”, without the chromophores inserted) and, remarkably, we have observed a dramatic stabilization of the protein with the chromophores bound. For instance, the melting temperature (temperature at which half of the proteins within the sample ensemble are folded and half are unfolded) for Alpha increases from 37°C to 79°C, and for Beta from 50°C to 67°C, whereas for Epsilon (a control protein that cannot bind chromophores) the melting temperature does not change when chromophores are added to the solution, demonstrating that the thermal stabilization is promoted by the presence of the chromophores inside the protein. We are writing two manuscripts based on these results.
Aim 2 has not been started yet (it requires Aim 1 to be completed).
Regarding Aim 3: In parallel to Aims 1-2, this Aim intends to advance the understanding of the role of coherence in enhancing function in photosynthesis, that is, LH and CS in natural pigment-protein complexes. We have successfully performed Two-Dimensional Electronic Spectroscopy on two LH complexes isolated from plants: CP43 and CP47. We have obtained high-quality spectroscopic data which we are carefully processed and analyzed. The results allow us to unravel multiple energy transfer pathways within CP43/CP47 with unprecedented detail, never before the energy transfer from each excitonic band to the lowest energy states in these systems could have been resolved. Even more exciting, we have been able to demonstrate that energy transfer in CP43/CP47 proceeds via a vibration-assisted (vibronic) mechanism, a highly debated topic in the literature. Finally, we have written a scientific manuscript based on these results.
On the Artificial Photosynthesis part of the project, the implementation of excitons into our chromophore-protein assemblies is a breakthrough that advances the artificial photosynthesis field considerably beyond the state of the art. Despite previous efforts and significant achievements in the field of protein design to build functional chromophore-protein systems, with remarkable successes attained by the scientific community, I believe that our approach towards the construction of robust and efficient light-harvesting and charge-separation units as described in my BioInspired_SolarH2 proposal is unique and will provide many more remarkable achievements that will significantly advance the field in the next months/years, that will result in several manuscripts that will be published in high impact journals. More specifically, I consider our approach unique because of the combination of: knowledge about the design principles of photosynthesis (I do have first-hand knowledge about how these principles work because I have been studying them extensively in previous years), a controllable and flexible system (the chromophore-protein assemblies have the potential to be optimized gradually as we obtain more information about their structural, energetic and dynamical properties by our spectroscopic methods), a collection of complementary spectroscopic methods [steady-state: Absorption, Fluorescence, Linear and Circular Dichroism, Stark, Raman, FTIR, Fluorescence Line-Narrowing; and time-resolved: Two-Dimensional Electronic Spectroscopy (2DES), Transient Absorption, Time-correlated Single Photon Counting (TCSPC)] that provide a complete physical picture of our systems under study. In addition to that, I count with a vast network of collaborators that can provide specific samples such as genetically-modified and natural pigment-protein assemblies, as well as knowledge about protein design and state-of-the-art spectroscopic methods.