Microbes in Ice Yield Potential Brain Control Molecules

Discovery may lead to advanced tools for manipulating brain cell activity using light.

Imagine glaciers, snow-capped mountains, and ice-cold groundwater—these frigid environments are home to unique molecules with the potential to manipulate brain cell activity. Structural biologist Kirill Kovalev is studying these molecules, called rhodopsins, for their potential use in optogenetics.

New Family of Rhodopsins

Kovalev, an EIPOD Postdoctoral Fellow at EMBL Hamburg’s Schneider Group and EMBL-EBI’s Bateman Group, is captivated by rhodopsins, colorful proteins that enable aquatic microorganisms to harness sunlight for energy. He is searching for unusual types to understand their undiscovered functions and potential benefits.

“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.”

Optogenetics and Cryorhodopsins

Some rhodopsins have been adapted as light-operated switches for cells’ electrical activity. This technique, optogenetics, allows neuroscientists to control neuronal activity during experiments. Rhodopsins with enzymatic activity could control chemical reactions using light. Recently, scientists have also started exploring the use of optogenetics to restore sensory functions in people with disabilities, such as hearing loss (National Institutes of Health 2021).

Kovalev discovered a new group of rhodopsins unlike any he had seen before, found exclusively in very cold environments. He named them ‘cryorhodopsins’.

Cryorhodopsins are a group of proteins found in cold-loving microorganisms. They have the remarkable ability to turn cellular electrical activity on and off Credit Daniela Velasco/EMBL

The Color Key

Color is a key feature of rhodopsins. Most are pink-orange, reflecting pink and orange light while absorbing green and blue, which activates them. Scientists aim to create differently colored rhodopsins to control neuronal activity with greater precision.

To Kovalev’s surprise, the cryorhodopsins he examined showed diverse colors, including blue ones.

“I can actually tell what’s going on with cryorhodopsin simply by looking at its colour,” laughed Kovalev.

Using advanced structural biology techniques, he discovered that the blue color is due to a rare structural feature. This discovery allows the design of synthetic blue rhodopsins for different applications.

Phylogeny of CryoRs. (A) Maximum likelihood phylogenetic tree of MRs. The tree includes 2199 sequences reported in (1), 3 sequences of DSE rhodopsins reported in (17), and 40 sequences of CryoRs found in the present work. (B) Enlarged view of the tree branch containing CryoRs. Amino acid residues in helix B at the position corresponding to that of T46 in BR are shown at the tips. (C) Rectangular representation of the phylogenetic tree of the CryoRs and nearby DSE and ACI rhodopsins clades. The inset in the left bottom corner shows amino acids of the seven-letter motifs of CryoR1-5, DSE, and ACI rhodopsins (numbering corresponds to CryoR1). The unique arginine (R57 in CryoR1) is boxed for clarity. — Science Advances

Switching Cells On and Off

Collaborators examined cryorhodopsins in cultured brain cells. Exposure to UV light induced electric currents. Green light increased cell excitability, while UV/red light reduced it.

“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. “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.

UV Light Protection

Cryorhodopsins can sense UV light, even on a rainy day, and respond slowly. This suggests they might act as photosensors, allowing microbes to ‘see’ UV light. Kovalev wondered if cryorhodopsins team up with a messenger molecule.

Working with collaborators from Alicante, Spain, and his EIPOD co-supervisor, Alex Bateman from EMBL-EBI, he noticed that the cryorhodopsin gene is accompanied by a gene encoding a small protein of unknown function, likely linked.

Using the AI tool AlphaFold, the team showed that five copies of the small protein could form a ring and interact with the cryorhodopsin, carrying information into the cell upon UV light detection.

Kovalev suspects that cryorhodopsins evolved their unique features to help microbes sense UV light in cold environments like mountain tops, where UV radiation is intense.

“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!”

Revealing cryorhodopsins’ biology required overcoming technical challenges. Kovalev applied a 4D structural biology approach, combining X-ray crystallography at EMBL Hamburg beamline P14 and cryo-electron microscopy (cryo-EM) 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.”