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

Proteins are fundamental building blocks of life; understanding their function is key to understanding biological processes. Recent EPR structural analyses of proteins, including Gd-DEER and nitroxide rapid freeze-quench techniques, provide static distance distributions at various times after activation [1]. However, an in-depth functional understanding of proteins requires a technique for tracking their inter-residue movement in real time. Such techniques exist, and include time-resolved (tr) X-ray spectroscopy, tr IR spectroscopy, tr NMR, and Förster resonance energy transfer, though each presents challenges when working with light-activated proteins in vitro. To mitigate these challenges, we present rapidscan-enabled, high-field, 240 GHz Gd-Gd tr EPR (TiGGER) and demonstrate it on AsLOV2, a light-activated protein found in oats. It had been established that upon 450 nm illumination in solution, AsLOV2’s J?-helix unfolds and becomes disordered, though there were not direct measurements of this motion, until now. The mechanical relaxation time constants recorded by TiGGER and UV-Vis spectroscopy are similar, confirming a relationship between photoswitching of the chromophore and mechanical action [2].

[1] Potapov and Goldfarb, Appl Magn Reson, Jan. 2010.

[2] Maity*, Price*, et al., bioRxiv, Oct. 2022.