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In recent years, photoactive proteins such as rhodopsins have become a common target for cutting-edge research in the field of optogenetics. Alongside wet-lab research, computational methods are also developing rapidly to provide the necessary tools to analyze and rationalize experimental results and, most of all, drive the design of novel systems. The Automatic Rhodopsin Modeling (ARM) protocol is focused on providing exactly the necessary computational tools to study rhodopsins, those being either natural or resulting from mutations. The code has evolved along the years to finally provide results that arereproducibleby any user,accurateandreliableso as to replicate experimental trends. Furthermore, the code isefficientin terms of necessary computing resources and time, andscalablein terms of both number of concurrent calculations as well as features. In this review, we will show how the code underlying ARM achieved each of these properties.

 

This perspective article highlights the challenges in the theoretical description of photoreceptor proteins using multiscale modeling, as discussed at the CECAM workshop in Tel Aviv, Israel. The participants have identified grand challenges and discussed the development of new tools to address them. Recent progress in understanding representative proteins such as green fluorescent protein, photoactive yellow protein, phytochrome, and rhodopsin is presented, along with methodological developments.

 

Abstract The understanding of how the rhodopsin sequence can be modified to exactly modulate the spectroscopic properties of its retinal chromophore, is a prerequisite for the rational design of more effective optogenetic tools. One key problem is that of establishing the rules to be satisfied for achieving highly fluorescent rhodopsins with a near infrared absorption. In the present paper we use multi-configurational quantum chemistry to construct a computer model of a recently discovered natural rhodopsin, Neorhodopsin, displaying exactly such properties. We show that the model, that successfully replicates the relevant experimental observables, unveils a geometrical and electronic structure of the chromophore featuring a highly diffuse charge distribution along its conjugated chain. The same model reveals that a charge confinement process occurring along the chromophore excited state isomerization coordinate, is the primary cause of the observed fluorescence enhancement. 

Abstract The lack of a theory capable of connecting the amino acid sequence of a light-absorbing protein with its fluorescence brightness is hampering the development of tools for understanding neuronal communications. Here we demonstrate that a theory can be established by constructing quantum chemical models of a set of Archaerhodopsin reporters in their electronically excited state. We found that the experimentally observed increase in fluorescence quantum yield is proportional to the computed decrease in energy difference between the fluorescent state and a nearby photoisomerization channel leading to an exotic diradical of the protein chromophore. This finding will ultimately support the development of technologies for searching novel fluorescent rhodopsin variants and unveil electrostatic changes that make light emission brighter and brighter. 

We present a computational protocol for the fast and automated screening of excited-state hybrid quantum mechanics/molecular mechanics (QM/MM) models of rhodopsins to be used as fluorescent probes based on the automatic rhodopsin modeling protocol (a-ARM). Such “a-ARM fluorescence screening protocol” is implemented through a general Python-based driver, PyARM, that is also proposed here. The implementation and performance of the protocol are benchmarked using different sets of rhodopsin variants whose absorption and, more relevantly, emission spectra have been experimentally assessed. We show that, despite important limitations that make unsafe to use it as a black-box tool, the protocol reproduces the observed trends in fluorescence and it is capable of selecting novel potentially fluorescent rhodopsins. We also show that the protocol can be used in mechanistic investigations to discern fluorescence enhancement effects associated with a near degeneracy of the S1/S2 states or, alternatively, with a barrier generated via coupling of the S0/S1 wave functions. 

The concerted interplay between reactive nuclear and electronic motions in molecules actuates chemistry. Here, we demonstrate that out-of-plane torsional deformation and vibrational excitation of stretching motions in the electronic ground state modulate the charge-density distribution in a donor-bridge-acceptor molecule in solution. The vibrationally-induced change, visualised by transient absorption spectroscopy with a mid-infrared pump and a visible probe, is mechanistically resolved by ab initio molecular dynamics simulations. Mapping the potential energy landscape attributes the observed charge-coupled coherent nuclear motions to the population of the initial segment of a double-bond isomerization channel, also seen in biological molecules. Our results illustrate the pivotal role of pre-twisted molecular geometries in enhancing the transfer of vibrational energy to specific molecular modes, prior to thermal redistribution. This motivates the search for synthetic strategies towards achieving potentially new infrared-mediated chemistry. 

Abstract Color tuning in animal and microbial rhodopsins has attracted the interest of many researchers, as the color of their common retinal chromophores is modulated by the amino acid residues forming the chromophore cavity. Critical cavity amino acid residues are often called “color switches”, as the rhodopsin color is effectively tuned through their substitution. Well-known color switches are the L/Q and A/TS switches located in the C and G helices of the microbial rhodopsin structure respectively. Recently, we reported on a third G/P switch located in the F helix of the light-driven sodium pumps of KR2 and Js NaR causing substantial spectral red-shifts in the latter with respect to the former. In order to investigate the molecular-level mechanism driving such switching function, here we present an exhaustive mutation, spectroscopic and computational investigation of the P219X mutant set of KR2. To do so, we study the changes in the absorption band of the 19 possible mutants and construct, semi-automatically, the corresponding hybrid quantum mechanics/molecular mechanics models. We found that the P219X feature a red-shifted light absorption with the only exception of P219R. The analysis of the corresponding models indicate that the G/P switch induces red-shifting variations via electrostatic interactions, while replacement-induced chromophore geometrical (steric) distortions play a minor role. However, the same analysis indicates that the P219R blue-shifted variant has a more complex origin involving both electrostatic and steric changes accompanied by protonation state and hydrogen bond networks modifications. These results make it difficult to extract simple rules or formulate theories for predicting how a switch operates without considering the atomistic details and environmental consequences of the side chain replacement.