Development of Photoswitchable, Phase Separable Biomaterials

Phase separation is a highly dynamic process that leads to the formation of biomolecular condensates in cells, which compartmentalize the cell's cytoplasm. These condensates contain proteins with high concentrations of intrinsically disordered regions (IDR), which lack a fixed three-dimensional structure. We aim to reversibly control phase separation in human cells using the LOV domain, a protein sensor that homodimerizes when exposed to blue light, providing a platform for photoswitchable control of cellular dynamics. We hypothesize that extant proteins containing LOV domains may show some nontrivial association with intrinsically disordered regions, and that these proteins with IDR domains may reversibly induce phase separation under blue light activation in vitro. To determine intrinsically-disordered character, we processed sequences from a database of known LOV-containing proteins through the IUPRED2A algorithm, a web-based tool that provides probability scores for likely intrinsically disordered amino acids in a given sequence. The frequency in occurrence of these LOV-containing proteins as a function of IDR percentage showed normal distributions significantly different from that of randomly generated sequences that we processed through IUPRED2A. We expressed the highest scoring proteins in mammalian cells and monitored their ability to form condensates in the presence of blue light. These novel proteins will provide a new tool for researchers to better understand the phenomenon of phase separation in cells and its effects in vitro.