Comparative Studies of Lov Switching Mechanisms and Characteristics

Environmental cues regulate many biological processes, coordinating cellular pathways to respond to changing conditions. Such regulation is often initiated by sensory protein domains which expand their chemical repertoire by using small molecule ligands to convert environmentally-triggered changes into altered protein/protein interactions. Light-Oxygen-Voltage (LOV) domains present outstanding model systems for such processes, given their abilities to convert blue light into photochemically-driven formations of protein/flavin adducts and subsequent triggering of the allosteric control of effectors as diverse as kinases to DNA-binding domains. Using a combination of biophysics, biochemistry and cell biology, we seek to gain insight into the mechanistic controls of such environmental sensing domains for both fundamental understanding and subsequent artificial control.

To address these questions, we combine approaches from: 1). biophysics, including NMR and EPR spectroscopies plus HDX-MS to monitor conformation and dynamics, 2). In vitro biochemistry studies of function, and 3). Cell-based examination of function in complex settings. Integrated data from these methods across LOV-HTH, LOV-HK and RGS-LOV-DUF settings will be discussed. This information has shown several commonalities of LOV signaling in diverse classes of sensory proteins, and further, develop structure-based mutations to shift these proteins among these states. In particular, we note substantial differences in the degree of conformational rearrangements observed among photoactivation among different LOV proteins. Further, we also observe a range of difference in off kinetics among proteins under study, often with substantial differences in the timing of adduct breakage and functional deactivation. We have taken advantage of this mechanistic understanding to develop the artificial regulation of such systems, both in vitro and in living cells.

Taken together, our work integrates information across a fascinating class of natural switches and suggests routes by which these can be manipulated to achieve a wide range of technical outcomes.