Overview:
We have successfully modelled an atomistic structure of three photo-intermediates, as well as the transition mechanism between them. We have generated a new set of force-field parameters to simulate the retinal chromophore in ChR2 based on hybrid quantum-mechanics/molecular-mechanics (QM/MM) calculations. We have characterized the intermediates by calculating their spectroscopic properties, chemical shift, pore hydration and conductance. We have validated our structures by comparison with experimental observables such as FTIR spectroscopy, UV-Vis or EM. We have also aided our experimental collaborators in the structural characterization of non-natural chromophores in the active site of ChR2.
Given the lack of structural experimental data, our theoretical approach required a careful cycle of modelling and validation of each of the photo-intermediates as well as the engineered ChR2. Starting from the crystal structure of a chimeric channelrhodopsin, we modeled the closed (inactive) state of the channel. We validated the structure by calculating the spectroscopic properties and comparing them to the experimental observations. We found that the active has some flexibility that was not observed in the crystal structure (because it gives a static picture only of the protein) but that can explain the features of the absorption spectra. From this structure and using enhanced sampling methods, we modeled and validated two other photo-intermediates. We found the formation of a water pore and the mechanism of proton transfer. Our simulations also provided an atomistic explanation for FTIR experiments.
The classical force-field we developed reproduces accurately the same structure of the chromophore and the same protein-ligand interactions as the QM/MM trajectory, and is a remarkable improvement in reproducing the experimental data compared to the standard set of parameters. We have shown that we can further exploit these parameters to simulate all photo-intermediates and even different ligands with little modifications. In a multidisciplinary collaboration with the group of Prof. Bamberg, we aid in the design of a modified chromophore that shows interesting spectroscopic properties. We are currently using the methodological setup, the conformational ensembles and the force-field parameters to design new mutants with tuned biophysical properties.
