Triggered functional dynamics of AsLOV2 by time-resolved electron paramagnetic resonance at high magnetic fields

Abstract

We present time-resolved Gd-Gd electron paramagnetic resonance (TiGGER) at 240 GHz for tracking inter-residue distances during a protein's mechanical cycle in solution state. TiGGER makes use of Gd-sTPATCN as spin labels, whose favorable qualities include a spin-7 / 2 EPR-active center, a short linker, a narrow intrinsic linewidth, and virtually no anisotropy at high magnetic fields (8.6 T) when compared to nitroxide spin labels. Using TiGGER, we are able to determine that upon light activation, the J α -helix and N-terminus of AsLOV2 separate in less than 1 s and relax back to equilibrium with a time constant of approximately 60 s. TiGGER reveals that the light-activated longrange mechanical motion is slowed in the Q513A variant of AsLOV2 and is correlated to the similarly slowed relaxation of the optically excited chromophore as described in recent literature. Our results demonstrate that TiGGER has the potential to valuably complement existing methods for the study of triggered functional dynamics in proteins.