Light triggered functional dynamics of AsLOV2 by time-resolved EPR at high magnetic fields

Proteins are molecular machines that power life. To understand their operation as machines it is necessary to investigate the evolution of their conformational changes in time, triggered by an external stimulus. We chose to focus on the photoswitchable protein AsLOV2. The current state of understanding is that the structures of the dark and the lit states exist but the non-equilibrium trajectories that connect the states are unknown. The main impact of this knowledge gap is felt in the optogenetics community where there is a significant demand for improved light-driven actuators. To guide knowledge-driven engineering of LOV proteins, measurements of time-resolved structural changes are needed. We present a unique method based on the remarkable EPR properties of Gd-sTPATCN used as a spin-label of AsLOV2. At 8.6 Tesla, the EPR spectrum is dominated by a single sharp line<5G, making it exquisitely sensitive to dipolar broadening by the magnetic field of nearby Gd(III) metal centers.Tracking the EPR lineshape as a function of time, we successfully detect that light activation causes an increase in distance between the Aα; and Jα; helices of AsLOV2 in the solution state. We also resolved the importance of Q513 residue in coupling light to mechanical motion in AsLOV2, which has been a topic of debate for a long time. We observed light-triggered slowed down mechanical motion between Aα; and Jα; helices even on the mutation of Q513 to non-polar alanine, signifying that other factors or residues also play a major role in the actuation of mechanical motion in AsLOV2.Next, we plan to film a “molecular movie” of AsLOV2 by measuring inter-spin inter-residue distances using rapid-scan transient cwEPR in the solution state. We acknowledge financial support from NSF MCB-2025860 and UCOP through MRI-19-601107.