Time-Resolved Ion Mobility Mass Spectrometry to Solve Conformational Changes in a Cryptochrome

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Protein conformational changes are vital for biological functions.
• Understanding protein dynamics requires advanced structural determination methods.
• Cryptochromes are blue light photoreceptors with flexible C-terminal extensions (CTE) whose structure-function relationship remains unclear.

Purpose of the Study:

• To introduce and validate a novel time-resolved ion mobility mass spectrometry approach.
• To investigate the role of specific amino acids in the conformational dynamics of *Chlamydomonas reinhardtii* animal-like cryptochrome (Cr aCRY) upon blue light activation.
• To elucidate the function of the C-terminal domain and its helix α22 in light-induced structural transitions.

Main Methods:

• Development of time-resolved ion mobility mass spectrometry with blue light irradiation.
• Application of the method to a simple photoreceptor model and the *Cr*aCRY system.
• Analysis of mass and ion mobility spectra to monitor structural changes.

Main Results:

• The study successfully applied time-resolved ion mobility mass spectrometry to *Cr*aCRY.
• Aspartate 321 (D321) was identified as essential for triggering large-scale conformational changes in helix α22 and the CTE in the lit state.
• Aspartate 323 (D323) was found to influence the timing of these conformational changes.

Conclusions:

• The novel time-resolved ion mobility mass spectrometry method enables the study of rapid protein structural dynamics.
• Specific amino acid residues, D321 and D323, play critical roles in the blue light-induced conformational transitions of *Cr*aCRY.
• These findings provide insights into the mechanism of cryptochrome function and light-sensing.