Femtosecond Crystallography Reveals Cryptochrome Mechanisms

Sunlight drives many biological processes on Earth. Most living organisms use light-sensitive proteins called cryptochromes to align their internal clocks with the natural day-night cycle.

Professor Manuel Maestre-Reyes’ research group at NTU’s Department of Chemistry used time-resolved serial femtosecond crystallography to create a 3D molecular timeline of cryptochrome activation. Their data captured structural changes occurring between 10 nanoseconds and 233 milliseconds after light exposure.

The study shows that cryptochrome acts as a signal amplifier. After detecting light, it undergoes rapid, subtle changes in its flavin adenine dinucleotide (FAD) chromophore. These changes trigger slower and more pronounced shifts in the protein's structure.

In the first step, the protein photoreduces FAD, forming a radical pair (FAD•–/Y373• RP). This pair activates three regions of the protein: the FAD binding site, the transient protonation pathway (TPP), and helix α22 near the Y373 residue.

Within nanoseconds, the FAD binding site stabilizes the radical pair by forming a hydrogen bond with FAD•–. This hydrogen bond rearrangement then activates the TPP within microseconds. The TPP extends the life of the radical pair into the millisecond range by providing a proton to FAD•–.

Moments later, the nearby presence of Y373• causes helix α22 to unfold like a ribbon. This structural change signals that cryptochrome has detected light.

These results clarify how light triggers molecular changes in cryptochromes. Temporary radical pair formation and charge separation, as observed in this process, also play key roles in oxidative phosphorylation, photosynthesis, and possibly in magnetic sensing. As such, cryptochrome may serve as a useful model for studying these broader processes in biophysical chemistry.