Bringing molecular pathways of rhodopsin fluorescence to light

Researchers have long sought to understand the black box of the brain. A group of proteins called microbial rhodopsins can help investigations of neuron-neuron signal transmission. When scientists edit neurons to express rhodopsins, the neurons become light-sensitive, enabling triggering, silencing, and signal visualization as they fluoresce. Further probing neural signaling requires engineering stronger fluorescing rhodopsins.

Walisinghe et al. constructed computer models of rhodopsins to tune their light absorption and emission at different intensities and colors. They found that the topography of rhodopsins’ internal electrostatic field was the most important factor for fluorescence.

“There is no law, there are no rules to really find the correct combination of amino acids to get certain properties,” said author Massimo Olivucci. “Without this guidance, [researchers are] just randomly mutating the proteins with the hope that maybe by luck they get the right combination.”

The researchers first built a computer model of Archaearhodopsin-3 for tuning light absorption and emission. Then, they mimicked mutations by virtually replacing different amino acids until they observed the intended fluorescent properties and could develop a mechanistic theory based on electrostatic potential topographies.

While the protein’s electrostatic field strongly determined the intensity and color of fluorescence, the protein cavity shape mattered, too. That’s because the light-emitting molecule, a derivative of Vitamin A called retinal chromophore, lives within the cavity.

“[Biochemists] can use this theory to design new mutants that may have better properties to study brain function at the single neuron level,” said Olivucci.

In time, the researchers hope to probe other rhodopsin properties with the same model.

“This is just a basis of what could be a more complete theory,” said Olivucci.

Source: “Theoretical principles for designing fluorescent rhodopsins: Electrostatic control of excited-state pathways,” by Danushka Walisinghe, Filippo Sacchetta, and Massimo Olivucci, Chemical Physics Reviews (2026). The article can be accessed at https://doi.org/10.1063/5.0339458 .