Triggered Functional Dynamics of AsLOV2 by Time-Resolved Electron Paramagnetic Resonance at High Magnetic Fields

Area of Science:

• Biophysics
• Structural Biology
• Spectroscopy

Background:

• Proteins undergo mechanical cycles crucial for function.
• Tracking these dynamics in solution is challenging.
• Existing methods have limitations in time resolution and scope.

Purpose of the Study:

• To introduce time-resolved Gd-Gd electron paramagnetic resonance (TiGGER) for protein dynamics.
• To measure inter-residue distances during a protein's mechanical cycle in solution.
• To investigate the light-induced conformational changes in AsLOV2.

Main Methods:

• Utilized TiGGER at 240 GHz, employing Gd-sTPATCN spin labels.
• Gd-sTPATCN offers a spin-7/2 EPR-active center with minimal anisotropy at high fields (8.6 T).
• Compared TiGGER with traditional nitroxide spin labels.

Main Results:

• Demonstrated that AsLOV2's C-terminus and N-terminus separate in <1 second upon light activation.
• Observed relaxation back to equilibrium with a time constant of ~60 seconds.
• Showed that the Q513A variant of AsLOV2 exhibits slowed light-activated motion, correlating with chromophore relaxation.

Conclusions:

• TiGGER is a powerful new method for tracking protein dynamics in solution.
• The study provides insights into the mechanical cycle of AsLOV2.
• TiGGER complements existing techniques for studying triggered protein dynamics.