Left: The Time-resolved serial femtosecond crystallography experiment (TR-SFX). In TR-SFX, cryptochrome crystals are first activated by a blue pump laser, and then probed with the X-ray Free Electron laser (XFEL). Here the pump laser works like a film clapper, starting the action. Meanwhile the XFEL works like a camera, collecting a single data point at a time determined by the delay between the pump and the XFEL. To construct a single frame in the molecular movie, data from about 50000 crystals is required. Right: Assembling the 3D molecular movie. By changing the delay between pump and XFEL, each frame can be constructed and assembled into a movie describing how the small light signal (orange arrow) is amplified by the cryptochrome over time (orange star). Such a movie may require data from several million crystals.
Cryptochromes are light-sensitive proteins found in most living things, including plants and animals. They help them 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 (SACLA) in Japan initially. They collected nineteen individual “frames” spanning from 10 nanoseconds to 233 milliseconds after illumination to put together the final movie.
The resulting ultra-slow motion, atomic resolution film 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.
First, 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 state.
Within nanoseconds, 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.
In addition, because transient formation of radical pairs similar to those found in the cryptochrome photocycle also take part in energy production in cells, photosynthesis, and even magnetic field sensing by living beings. Thus, the cryptochrome molecular mechanism may act as a model system to better understand the fundamentals of these wide-ranging topics.
“With time-resolved crystallography, we have ensued in an age of protein movie making, giving unprecedented insight into the chemical principles of light-triggered processes in nature,” said Prof. Manuel Maestre-Reyna. “I am happy to represent the NTU at the forefront of this scientific frontier.”
Prof. Manuel Maestre-Reyna’s email address: [email protected]
