The TR-icOS setup at the ESRF: time-resolved microsecond UV-Vis absorption spectroscopy on protein crystals

Area of Science:

• Structural Biology
• Spectroscopy
• Biophysics

Background:

• Time-resolved macromolecular crystallography (TR-MX) is advancing with dedicated synchrotron beamlines.
• Microsecond time-resolved mechanistic studies of photoactive proteins are now feasible.
• Time-resolved spectroscopic experiments on protein crystals are crucial for determining optimal time delays.

Purpose of the Study:

• To develop and validate a setup for time-resolved spectroscopic analysis of single protein crystals.
• To establish experimental parameters for time-resolved macromolecular crystallography (TR-MX).
• To investigate laser-induced artifacts in photoreaction studies.

Main Methods:

• Construction of the time-resolved in crystallo Optical Spectroscopy (TR-icOS) setup at the European Synchrotron Radiation Facility.
• Utilizing a nanosecond laser as a pump and a microsecond xenon flash lamp as a probe.
• Recording pump-probe spectra from single crystals with time delays from microseconds to seconds.
• Performing power titrations by varying laser pulse energies to detect two-photon absorption artifacts.

Main Results:

• The TR-icOS setup allows rapid recording of pump-probe spectra from single crystals.
• The photocycle of bacteriorhodopsin was monitored, showing alterations at laser fluences exceeding 100 mJ/cm².
• Experimental parameters for laser pulse energy and delay were determined for successful TR-MX.

Conclusions:

• The TR-icOS instrument provides essential data for optimizing TR-MX experiments.
• Understanding laser-induced artifacts is critical for accurate mechanistic studies of photoreactions.
• This approach facilitates the study of photoactive proteins and other systems using TR-MX.