Kovalev, EIPOD Postdoctoral Fellow at EMBL Hamburg's Schneider Group and EMBL-EBI's Bateman Group, is a physicist passionate about solving biological problems. He is particularly hooked by rhodopsins, a group of colorful proteins that enable aquatic microorganisms to harness sunlight for energy.

"In my work, I search for unusual rhodopsins and try to understand what they do," said Kovalev. "Such molecules could have undiscovered functions that we could benefit from."

Some rhodopsins have already been modified to serve as light-operated switches for electrical activity in cells. This technique, called optogenetics, is used by neuroscientists to selectively control neuronal activity during experiments. Rhodopsins with other abilities, such as enzymatic activity, could be used to control chemical reactions with light, for example.

Having studied rhodopsins for years, Kovalev thought he knew them inside out - until he discovered a new, obscure group of rhodopsins that were unlike anything he had seen before.

As it often happens in science, it started serendipitously. While browsing online protein databases, Kovalev spotted an unusual feature common to microbial rhodopsins found exclusively in very cold environments, such as glaciers and high mountains. "That's weird," he thought. After all, rhodopsins are something you typically find in seas and lakes.

These cold-climate rhodopsins were almost identical to each other, even though they evolved thousands of kilometres apart. This couldn't be a coincidence. They must be essential for surviving in the cold, concluded Kovalev, and to acknowledge this, he named them 'cryorhodopsins'.

Rhodopsins out of the blue

Kovalev wanted to know more: what these rhodopsins look like, how they work, and, in particular, what color they are.

Color is the key feature of each rhodopsin. Most are pink-orange - they reflect pink and orange light, and absorb green and blue light, which activates them. Scientists strive to create a palette of different colored rhodopsins, so they could control neuronal activity with more precision. Blue rhodopsins have been especially sought-after because they are activated by red light, which penetrates tissues more deeply and non-invasively.

To Kovalev's amazement, the cryorhodopsins he examined in the lab revealed an unexpected diversity of colors, and, most importantly, some were blue.

The color of each rhodopsin is determined by its molecular structure, which dictates the wavelengths of light it absorbs and reflects. Any changes in this structure can alter the color.

"I can actually tell what's going on with cryorhodopsin simply by looking at its color," laughed Kovalev.

Applying advanced structural biology techniques, he figured out that the secret to the blue color is the same rare structural feature that he originally spotted in the protein databases.

"Now that we understand what makes them blue, we can design synthetic blue rhodopsins tailored to different applications," said Kovalev.

Next, Kovalev's collaborators examined cryorhodopsins in cultured brain cells. When cells expressing cryorhodopsins were exposed to UV light, it induced electric currents inside them. Interestingly, if the researchers illuminated the cells right afterwards with green light, the cells became more excitable, whereas if they used UV/red light instead, it reduced the cells' excitability.

"New optogenetic tools to efficiently switch the cell's electric activity both 'on' and 'off' would be incredibly useful in research, biotechnology and medicine," said Tobias Moser, Group Leader at the University Medical Center Göttingen who participated in the study. "For example, in my group, we develop new optical cochlear implants for patients that can optogenetically restore hearing in patients. Developing the utility of such a multi-purpose rhodopsin for future applications is an important task for the next studies."

"Our cryorhodopsins aren't ready to be used as tools yet, but they're an excellent prototype. They have all the key features that, based on our findings, could be engineered to become more effective for optogenetics," said Kovalev.

Evolution's UV light protector

When exposed to sunlight even on a rainy winter day in Hamburg, cryorhodopsins can sense UV light, as shown using advanced spectroscopy by Kovalev's collaborators from Goethe University Frankfurt led by Josef Wachtveitl. Wachtveitl's team showed that cryorhodopsins are in fact the slowest among all rhodopsins in their response to light. This made the scientists suspect that those cryorhodopsins might act like photosensors letting the microbes 'see' UV light - a property unheard of among other cryorhodopsins.

"Can they really do that?" Kovalev kept asking himself. A typical sensor protein teams up with a messenger molecule that passes information from the cell membrane to the cell's inside.

Kovalev grew more convinced, when together with his collaborators from Alicante, Spain, and his EIPOD co-supervisor, Alex Bateman from EMBL-EBI, they noticed that the cryorhodopsin gene is always accompanied by a gene encoding a tiny protein of unknown function - likely inherited together, and possibly functionally linked.

Kovalev wondered if this might be the missing messenger. Using the AI tool AlphaFold, the team were able to show that five copies of the small protein would form a ring and interact with the cryorhodopsin. According to their predictions, the small protein sits poised against the cryorhodopsin inside the cell. They believe that when cryorhodopsin detects UV light, the small protein could depart to carry this information into the cell.

"It was fascinating to uncover a new mechanism via which the light-sensitive signal from cryorhodopsins could be passed on to other parts of the cell. It is always a thrill to learn what the functions are for uncharacterised proteins. In fact, we find these proteins also in organisms that do not contain cryorhodopsin, perhaps hinting at a much wider range of jobs for these proteins."

Why cryorhodopsins evolved their astonishing dual function - and why only in cold environments - remains a mystery.

"We suspect that cryorhodopsins evolved their unique features not because of the cold, but rather to let microbes sense UV light, which can be harmful to them," said Kovalev. "In cold environments, such as the top of a mountain, bacteria face intense UV radiation. Cryorhodopsins might help them sense it, so they could protect themselves. This hypothesis aligns well with our findings."

"Discovering extraordinary molecules like these wouldn't be possible without scientific expeditions to often remote locations, to study the adaptations of the organisms living there," added Kovalev. "We can learn so much from that!"

Unique approach to unique molecules

To reveal the fascinating biology of cryorhodopsins, Kovalev and his collaborators had to overcome several technical challenges.

One was that cryorhodopsins are nearly identical in structure, and even a slight change in the position of a single atom can result in different properties. Studying molecules at this level of detail requires going beyond standard experimental methods. Kovalev applied a 4D structural biology approach, combining X-ray crystallography at EMBL Hamburg beamline P14 and cryo-electron microscopy (cryo-EM) in the group of Albert Guskov in Groningen, Netherlands, with protein activation by light.

"I actually chose to do my postdoc at EMBL Hamburg, because of the unique beamline setup that made my project possible," said Kovalev. "The whole P14 beamline team worked together to tailor the setup to my experiments - I'm very grateful for their help."

Another challenge was that cryorhodopsins are extremely sensitive to light. For this reason, Kovalev's collaborators had to learn to work with the samples in almost complete darkness.

Using observations in mice, led primarily by first author and PhD student José Antonio González Parra and supervised by Dr. Arnau Busquets, the research team was able to determine the mechanisms involved in how the brain makes decisions based on indirect associations between different stimuli. That is, instead of directly associating a specific stimulus with a rewarding or aversive situation, the brain establishes connections between two or more stimuli. As Dr. Busquets explains, "The project aims to understand how the brain enables us to make decisions based on indirect relationships between stimuli in our environment."

In this context, the mice were subjected to various behavioral tests. They were trained to associate one smell-banana-with a sweet taste, and another smell-almond-with a salty taste. Later, a negative stimulus was associated with the smell of banana. From that point on, the mice rejected the sweet taste, which was linked to the banana smell and thus carried a negative connotation. In other words, "they formed an indirect association between the sweet taste and the aversive stimulus through its link to a specific smell," explains Busquets.

The Role of the Amygdala

Using genetic techniques delivered via viral vectors, the researchers were able to observe which areas of the mice's brains were activated throughout the process of encoding and consolidating the associations. They found that the amygdala, a brain region associated with responses such as fear and anxiety and involved in certain mental disorders like psychosis and PTSD, was activated when the mice linked olfactory and taste stimuli.

At the same time, they identified other brain areas that were also involved and interacted with the amygdala. Thanks to imaging techniques, they were able to establish a connection between these areas and a part of the cerebral cortex. "We have identified a brain circuit that controls associations between stimuli and allows for these indirect associations," says Dr. Busquets. They also confirmed that if amygdala activity was inhibited while the mice were exposed to the stimuli, the animals were unable to form these indirect associations.

As Dr. Arnau Busquets explains, the researchers believe that the brain circuits involved in decision-making processes in humans are similar to those in mice. Therefore, the data obtained in this newly published study could be relevant for treating certain mental disorders linked to amygdala activity. "Alterations in these indirect associations form the basis of various mental disorders," he adds. "Understanding the brain circuits involved in these complex cognitive processes can help us design therapeutic strategies for humans." In this sense, future approaches could include brain stimulation or modulation of activity in these areas in people with PTSD or psychotic symptoms.