Colouring cryo-cooled crystals: online microspectrophotometry

Area of Science:

• Biophysics
• Structural Biology
• Spectroscopy

Background:

• X-ray crystallography often uses cryo-cooling for macromolecular crystals.
• X-ray radiation can induce chemical changes, forming radicals within crystals.
• Radicals can have distinct spectral properties, enabling their detection.

Purpose of the Study:

• To characterize X-ray-induced radicals in cryo-cooled macromolecular crystals.
• To investigate the optical and spectral properties of these radicals.
• To develop a method for simultaneous X-ray data collection and spectral analysis.

Main Methods:

• Construction of an online microspectrophotometer with high temporal resolution.
• Measurement of UV/VIS absorption spectra (200-1100 nm) during X-ray data collection.
• Analysis of X-ray-induced spectral changes in cryo-cooled samples.

Main Results:

• Identification of X-ray-induced blue color as trapped solvated electrons.
• Detection of disulfide radicals (absorption maxima ~400 nm) in proteins.
• Observation of radical lifetimes in the range of seconds to minutes at 100 K.
• Different temperature-dependent kinetics for solvated electrons and disulfide radicals.

Conclusions:

• The developed microspectrophotometer is a valuable tool for studying transient species in macromolecular crystals.
• Solvated electrons and disulfide radicals are key X-ray-induced intermediates.
• This technique complements X-ray diffraction for analyzing protein redox states and intermediates.