Rare blue proteins from cold-adapted microbes could serve as prototypes for molecular on-off switches

Imagine the magnificent Greenland glaciers, eternal snow of high Tibetan mountains and icy groundwater in Finland. As cold and beautiful as, for the structural biologist Kirill Kovalev, they house more important molecules which could control the activity of brain cells.

Kovalev, a postdoctoral stock market Eipod of the Schneider group of emburg and the Bateman group of EMBL-EBI, is a physicist passionate about the solving biological problems. It is particularly hung by rhodopsins, a group of colorful proteins that allow aquatic microorganisms to exploit sunlight for energy.

“In my work, I’m looking for unusual rhodopsins and try to understand what they are doing,” Kovalev said. “Such molecules could have unknown functions that we could benefit from.”

Some rhodopsins have already been modified to serve as switches to the electrical activity of cells. This technique, called optogenetics, is used by neuroscientists to selectively control neural activity during experiences. Rhodopsins with other capacities, such as enzymatic activity, could be used to control chemical reactions with light, for example.

Having studied rhodopsins for years, Kovalev thought of knowing them – until he discovered a new obscure group of rhodopsins which did not resemble everything he had seen before.

As often happens in science, he started by chance. When browsing online protein databases, Kovalev identified an unusual characteristic common to microbial rhodopsins found exclusively in very cold environments, such as glaciers and high mountains.

“It’s weird,” he thought. After all, rhodopsins are something that you generally find in seas and lakes.

These cold climate rhodopsins were almost identical to each other, even if they have evolved thousands of kilometers. This could not be a coincidence. They must be essential to survive in the cold, concluded Kovalev, and to recognize it, he called them “cryorhodopsins”.

Unexpected rhodopsins

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

Color is the key characteristic 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 rhodopsins of different colors, so that they can control neural activity with more precision. Blue rhodopsins have been particularly sought after because they are activated by red light, which penetrates the tissues more deeply and in a non -invasive manner.

At the astonishment of Kovalev, the cryorhodopss he examined in the laboratory revealed an unexpected diversity of colors and, above all, were blue. The work is published in the journal Scientific advances.

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

“I can really say what’s going on with Cryorhodopsin simply by looking at his color,” said Kovalev.

By applying advanced structural biology techniques, he understood that blue color secret is the same rare structural characteristic which he initially identified in protein databases.

“Now that we understand what makes them blue, we can design synthetic blue rhodopsins adapted to different applications,” said Kovalev.

Then, Kovalev employees examined cryorhodopsins in cultivation cerebral cells. When the cells expressing cryorhodopsins were exposed to UV light, it induced electric currents inside. Interestingly, if the researchers lit up the cells just after with a green light, the cells have become more excitable, while if they used in UV / red place, this reduced the excitability of the cells.

“New optogenetic tools to effectively change the electrical activity of the cell both” on “and” off “would be incredibly useful in research, biotechnology and medicine,” said Tobias Moser, group leader of the University Medical Center Göttingen, who participated in the study.

“For example, in my group, we are developing new optical cochlear implants for patients who can restore optogenetically hearing in patients. Developing the usefulness of such a versatile rhodopsin for future applications is an important task for the next studies.”

“Our cryorhodopsins are not yet ready to be used as tools, but they are an excellent prototype. They have all the key characteristics which, on the basis of our results, could be designed to become more effective for optogenetics,” said Kovalev.

UV Light Protector of Evolution

When exposed to the sun very by a rainy winter day in Hamburg, cryorhodops can smell UV light, as shown by spectroscopy advanced by Kovalev collaborators from Goethe Frankfort University, led by Josef Wachtveitl.

The Wachtveitl team has shown that cryorhodopsins are in fact the slowest among all rhodopsins in their response to light. This has made scientists suspect that these cryorhodopsins could act as photosensions, leaving microbes “see” UV light – an unknown property among other cryorhodops.

“Can they really do that?” Kovalev kept wondering. A typical sensor protein is associated with a messenger molecule which transmits information from the cell membrane to the cell inside.

Kovalev became more convinced, when his collaborators of Alicante, Spain, and his Eipod co-supervisor, Alex Bateman of Embl-Ebi, have noticed that the cryorhodopsin gene is always accompanied by a gene coding for a tiny protein of unknown function-inherent together, and perhaps functionally linked.

Kovalev wondered if it could be the missing messenger. Using the AI ​​Alphafold tool, the team was able to show that five copies of the small protein would form a ring and interact with cryorhodopsin.

According to their predictions, the small protein is on cryorhodopsin inside the cell. They believe that when cryorhodopsin detects UV light, the small protein could leave to transport this information in the cell.

“It was fascinating to discover a new mechanism via which the signal sensitive to the light of cryorhodopsins could be transmitted to other parts of the cell. It is always a pleasure to learn what are the functions for not characterized proteins. In fact, we also find these proteins in organisms that do not contain Cryorhodopsin, perhaps winding in a much broader range of these proteins.” “”

Why cryorhodopsins have evolved their astonishing double function – and why in cold environments – remain a mystery.

“We suspect that cryorhodops have evolved their unique characteristics not because of the cold, but rather to let the microbes feel UV light, which can be harmful to them,” said Kovalev.

“In cold environments, like the top of a mountain, bacteria face intense UV radiation. Cryorhodopsins could help them detect it, so that they can protect themselves. This hypothesis aligns well with our results.”

“Discovering extraordinary molecules like these would not be possible without scientific expeditions to often distant places, to study the adaptations of the organizations that live there,” added Kovalev. “We can learn so much!”

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 almost identical in the structure, and even a slight change in the position of a single atom can lead to different properties. The study of molecules at this level of detail requires going beyond standard experimental methods.

Kovalev applied a 4D approach to structural biology, combining X-ray crystallography with the Hamburg P14 EMBL beam line and cryo-electron microscopy (cryo-EM) in the group of Albert Guskov in Groningen, Netherlands, with the activation of proteins by light.

“I actually chose to do my post-doctorator at EMBL Hamburg, due to the unique configuration of the beam line that made my project possible,” Kovalev said.

“The entire P14 Beamline team has worked together to adapt the configuration to my experiences – I am very grateful for their help.”

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