Discussion
In the design of optogenetic tools, a fundamental limitation of LOV-based systems is the lack of a digital switch from a non-functional off-state to a highly-function on-state. Rather, optogenetic tools have residual activity in both the Light and Dark states thereby limiting their fidelity. To examine the origins of the lack of a digital LOV-based photoswitch, we wished to computationally examine the allosteric activity of LOV based proteins to extract information relevant to the conformational landscape and configurational entropy of Dark and Light states of VVD. In so doing, we discovered an inherent difference in correlating sampling of conformational space with calculations of configurational entropy. Specifically, where the VVD Dark state samples more conformational space, but the Light state has higher configurational entropy. These analyses shed light on the nature of allostery in LOV based photoswitches.

Examination of the unperturbed 210 ns simulations revealed profound differences between the overall dynamics of VVD Dark and Led SMD Bulbs states. Specifically, the Dark state samples more conformational space during the course of 210 ns simulations than the Light state (Fig. 1B). Further, the Dark state samples conformational space, which lie close to the Light state, thereby likely contributing to residual activity in both states and loss of a digital switch as observed in optogenetic tools. The counterintuitive nature of the conformational sampling of the Dark state, could lie in the need to facilitate a fast switch from the dormant Dark state to active Light state and to reduce barriers in the transition. In such a system, the Dark state samples conformational space poised for activity. Blue-light then populates the Light state that is “locked” into specific conformational space to better serve its signaling function.