Scientists unveil the mechanism of light-sensing proteins that regulate internal clock

Cryptochromes are light-sensitive proteins found in all living things, including plants and animals. They help living things keep time with day and night by controlling internal clocks and responses to light. They are important for things like sleep cycles, plant growth, and possibly even sensing Earth’s magnetic field.

Until now, scientists knew that cryptochromes function by absorbing blue light, which triggers structural changes in the protein. This activates interactions with other cellular proteins, influencing gene expression and biological rhythms. However, the mechanism by which cryptochromes manifest this light-sensing ability remained unclear.

A team led by Manuel Maestre-Reyna at the National Taiwan University has now filmed a high-resolution, 3D molecular movie of a cryptochrome in action.

To achieve that, the team used time-resolved serial femtosecond crystallography (TR-SFX) at Spring-8 Angstrom Compact X-ray Free Electron Laser in Japan initially. They collected nineteen individual “frames” spanning from 10 nanoseconds to 233 milliseconds after illumination to put together the final movie.

TR-icOS completes the picture

They then used transient absorption spectroscopy (TAS) at the TR-icOS instrument at the ESRF. Maestre-Reyna explains the importance of these experiments at the ESRF: “The role of the ESRF, and in particular of TR-icOS, was crucial. Without in crystallo TAS experiments, that currently can only be performed at TR-icOS, the biological relevance of our results would be highly questionable. Furthermore, TRX can only detect structural changes, and is limited by the data quality in its ability to do so. In other words, chemical transformations that imply only very subtle structural changes, such as electron transfer, cannot be easily tracked by TRX. On the contrary, TAS can very easily detect such electronic reconfigurations, which change protein colour. Only by combining the two was it possible to fully understand the molecular mechanism of electron-transfer based signaling of cryptochromes”.

The experiments resulted in an ultra-slow motion, atomic resolution film that explains how the cryptochrome protein amplifies the subtle photochemical signal, which then snowballs into dramatic structural changes. The process is coordinated by the protein, with three molecular regions acting in unison to accomplish sensing.

Specifically, during the initial photochemical change, flavin adenine dinucleotide (FAD), a special light-gathering moiety within the protein, used the energy of blue light to capture an electron from the cryptochrome itself, inducing a highly unstable radical pair (RP) state. Early on, the protein attempts to stabilize this short-lived species by modulating its immediate environment. These local changes cascade over time, until, by about 100 milliseconds after RP formation, entire regions of the protein unfold like a ribbon, signaling that cryptochrome has sensed light.

Understanding sleep disorders

The results of this study provide a detailed description of the molecular basis of cryptochrome function, which can be relevant in research related to circadian rhythm, such as that focused on sleep disorders.

For example, recent studies have linked mutations in human cryptochrome 2 (CRY2) to Advanced Sleep Phase Disorder (ASPD). This disorder is characterized by disruption of the circadian rhythm, as patients fall asleep by 7 pm, but wake up at ~2 am, and it is not treatable with melatonin nor other effective therapeutics.

“Whilst our research is very fundamental, we hope that by delineating the structural principles of cryptochrome we can maybe lead to new drug design for the modulation of CRY2”, concludes Maestre-Reyna.

Reference:

Maestre-Reyna, M. et al., Science Advances 2025. DOI: 10.1126/sciadv.adu7247

Text by Montserrat Capellas Espuny