Switchable rhodOpsins in Life Sciences

Project Information

SOL

Grant agreement ID: 951644

DOI

10.3030/951644

EC signature date 7 February 2021

Start date 1 September 2022

End date 31 August 2028

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 7 971 388,75

EU contribution

€ 7 971 388,75

Coordinated by

PAUL SCHERRER INSTITUT
Switzerland

Periodic Reporting for period 2 - SOL (Switchable rhodOpsins in Life Sciences)

Reporting period: 2024-03-01 to 2025-08-31

Summary of the context and overall objectives of the project

The SOL - Switchable rhodOpsins in Life Sciences – project aims to identify and characterise light-switchable proteins from nature and optimise them to become optogenetics tools for manipulating cellular function. The SOL project is based on bistable rhodopsins, which belong to the class of G protein coupled receptors (GPCRs). There are hundreds of different GPCRs activating a variety of different G proteins, and they play an important role in cell signalling in almost every cell type. Not surprisingly, they are the targets of a large variety of pharmaceuticals. Rhodopsins are light-activated GPCRs, best known for their role as light receptors in the retina of the human eye. Upon activation, the visual receptors in our eyes lose their light sensor (chromophore), the vitamin A derivative retinal, and it must be “reassembled” with the protein in order to accept photons (light) again. Bistable rhodopsins, however, keep their retinal and can be activated and deactivated by flashes of different light colours without requiring any assembly, acting as true biological “switches”. They will become revolutionary tools in physiology and neurobiology.
The SOL project tackles the limitations of current optogenetics tools, which primarily utilize light-gated ion channels, confining applications mainly to nerve cell stimulation. This lack of flexibility has hindered broader applications in life sciences, particularly regarding the control of GPCRs, which are crucial for cell signalling. OptoGPCRs will offer higher sensitivity and better spatial and temporal resolution.
Understanding and controlling GPCRs is vital because these receptors are implicated in numerous physiological processes and are targets for a wide array of pharmaceuticals. By developing bistable rhodopsins that act as light-activated biological switches, the project could revolutionize drug discovery. They allow a much better controlled target identification because they can be used to verify the signalling output of a receptor in vivo. This paves the way for advancements in gene therapeutics and improved treatments for various human diseases.
In the SOL project, we are to investigate the structure of bistable rhodopsins to understand their functionality better. We aim to engineer new bistable rhodopsins capable of being activated and deactivated by different wavelengths of light, effectively mimicking GPCR signaling. We apply these light-controlled switches to study G protein signalling effects in animal models. With this we aim to use this knowledge for developing optogenetics for understanding complex physiology and the brain. Another important application is engineering signalling circuit for hormone release.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Recent research efforts of the SOL consortium have significantly advanced the understanding and applications of bistable rhodopsins and GPCR signalling. We have emphasized the role of water-mediated hydrogen bond networks in determining the efficiency of G protein activation during signalling, shedding light on the intricate structural dynamics that influence receptor behaviour. In another notable study, we developed an application using an all-trans retinal analogue to promote specific states of bistable opsins, thereby enhancing the toolkit available for manipulating GPCR activity with light. This advancement also promotes our recent success in determining the active state structures of a bistable opsin bound to G proteins, effectively linking the photoswitchable properties of retinal to the water network that affects the spectral characteristics of the receptor. This pivotal work was accepted for publication in a prestigious journal, underscoring its significance.

Moreover, we explored the potential of the non-visual human pigment OPN5 as an optogenetics tool for Gq pathway control even in deep tissues, showcasing the future of targeted signalling while minimizing the side effects commonly associated with broader GPCR activation. Another analysis on the genetic evolution of rhodopsin cyclases in fungi contributes to a deeper understanding of light sensitivity adaptations within the realm of animal evolution.

The investigation on the electronic and geometrical structure of a near-infrared absorbing microbial rhodopsin reveals critical insights into charge distribution that influence its spectral properties. Additionally the complexities of photoactivation processes was clarified by discovering multiple retinal isomerization stages during the early phases of the bestrhodopsin photoreaction. Lastly, by utilizing terahertz Stark spectroscopy we reveal excited-state dipole moments in the retinal chromophore, shedding light on the mixing states that affect proton pumping efficiencies in bacteriorhodopsin. Collectively, these studies represent a significant progression in the field, paving the way for innovative applications in optogenetics and cellular signalling.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The progress made by our consortium has significantly advanced the field of optogenetics and GPCR signalling beyond the current state of the art. Our research has uncovered fundamental insights into the retinal isomerization of a novel protein -bestrhodopsin - using the most advanced spectroscopic methods available. These technologies are being directly applied to our novel optoGPCR targets, including the chimera constructs, which we have successfully developed.

We have also adapted time-resolved cellular assays initially developed for melanopsin to our bistable pigment targets such as invertebrate rhodopsins. These assays proved essential for characterizing an invertebrate rhodopsin mutant with good spectral separation, which will facilitate the characterization of all engineered bistable rhodopsins. This effort is crucial for the progress of the SOL project, and we are currently on track to meet our objectives.

A major milestone in our research is determining the structure of an active invertebrate rhodopsin. This breakthrough has deepened our understanding of the relationship between G proteins in invertebrates and vertebrates, leading to the development of a new tool for structural biology that enhances the efficiency of cryo-EM studies for bistable rhodopsins. Furthermore, we have explored the use of retinal analogues as super agonists, successfully integrating this strategy into our toolbox. Our structural work has led to immediate impacts on our protein engineering approach, resulting in the creation of a fusion protein to activate desired signalling pathways. We have combined colour-shifting mutations with GPCR fusions, yielding promising proof of bistability for this construct in vitro and in cellular assays.

Additionally, we have extended our strategy to include various constructs targeting cone pigments, alongside novel targets like insect rhodopsin and parapinopsins. Our research on crustacean rhodopsins has evolved from shrimp to well-expressing fish louse pigments, integrating the latest findings. In collaboration with Japanese teams, we have supported studies on coral opsins, helping to solidify proposal that chloride acts as a counterion.

We develop an AI approach for wavelength prediction for retinal proteins, which shows excellent potential for both microbial rhodopsins and bistable pigments. We have also employed QM/MM methods to calculate absorption maxima for visual pigments. In a remarkable simulation of polyene isomerization conical intersections, they provided detailed ab initio calculations that illustrated how the surrounding environment influences the reaction mechanism based on the shape of the conical intersection. Overall, this progress is expected to yield significant advancements in understanding and manipulating bistable rhodopsins and GPCR signalling, pushing the boundaries of optogenetics applications.