Slow protein dynamics probed by time-resolved oscillation crystallography at room temperature

Area of Science:

• Structural Biology
• Biophysics
• Crystallography

Background:

• Serial crystallography advances enable studying fast protein dynamics (picoseconds to seconds).
• Slower protein dynamics (seconds to hours) remain challenging to study structurally.
• Room-temperature macromolecular crystallography (RT-MX) offers potential for studying these slower dynamics.

Purpose of the Study:

• To structurally characterize the slow relaxation of a photoreaction intermediate in the LOV2 domain of phototropin 2 from Arabidopsis thaliana.
• To extend the capabilities of RT-MX to study protein dynamics on the second-to-hour timescale.

Main Methods:

• Utilized synchrotron-based RT-MX with a fast-readout detector (EIGER X 4M) to collect X-ray diffraction data.
• Employed *in crystallo* UV-Vis absorption spectroscopy to monitor the photoreaction and relaxation.
• Collected multiple diffraction datasets at various time points during the intermediate's decay.

Main Results:

• Successfully monitored the relaxation of a photoadduct intermediate, involving a thio-ether bond formation.
• Observed unexpected protein rearrangements and a crystal phase transition after the chromophore returned to its ground state.
• Found that these rearrangements hindered the full recovery of the protein's structural ground state.

Conclusions:

• The study demonstrates the feasibility of time-resolved protein crystallography at synchrotrons for slow dynamic processes (second-to-hour timescale).
• Revealed an unexpected role for a conserved tryptophan residue in regulating the LOV2 photocycle.
• Highlights that protein structural recovery may be incomplete due to light-induced rearrangements and crystal lattice changes.