Disruption of the FMN-A524 interaction cascade and Glu513-induced collapse of the hydrophobic barrier promotes light-induced Ja-helix unfolding in AsLOV2

The C-terminal Ja-helix of the Avena sativa's Light Oxygen and Voltage (AsLOV2) protein, unfolds on exposure to blue light. This characteristic seeks relevance in applications related to engineering novel biological photoswitches. Using molecular dynamics simulations and the Markov state modeling (MSM) approach we provide the mechanism that explains the stepwise unfolding of the Ja-helix. The unfolding was resolved into seven steps represented by the structurally distinguishable states distributed over the initiation and the post initiation phases. Whereas, the initiation phase occurs due to the collapse of the interaction cascade FMN-Q513-N492-L480-W491-Q479-V520-A524, the onset of the post initiation phase is marked by breaking of the hydrophobic interactions between the Ja-helix and the I0-strand. This study indicates that the displacement of N492 out of the FMN binding pocket, not necessarily requiring Q513, is essential for the initiation of the Ja-helix unfolding. Rather, the structural reorientation of Q513 activates the protein to cross the hydrophobic barrier and enter the post initiation phase. Similarly, the structural deviations in N482, rather than its integral role in unfolding, could enhance the unfolding rates. Furthermore, the MSM studies on the wild-type and the Q513 mutant, provide the spatiotemporal roadmap that lay out the possible pathways of structural transition between the dark and the light states of the protein. Overall, the study provides insights useful to enhance the performance of AsLOV2-based photoswitches. SIGNIFICANCE As a light-sensitive protein, AsLOV2 has been widely demonstrated for its potential for developing biological photoswitches. The mechanistic insights provided by this study will be important in designing novel and efficient photoswitches. Applications related to optogenetics and development of molecular tools in photobiology will benefit from this work.