Curated Optogenetic Publication Database

Abstract: Optogenetic tools for controlling protein-protein interactions (PPIs) have been developed from a small number of photosensory modules that respond to a limited selection of wavelengths. Cyanobacteriochrome (CBCR) GAF domain variants respond to an unmatched array of colors; however, their natural molecular mechanisms of action cannot easily be exploited for optogenetic control of PPIs. Here we developed bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s by engineering synthetic light-dependent interactors for a red/green GAF domain. The systematic approach enables the future engineering of the broad chromatic palette of CBCRs for optogenetics use. BICYCLs are among the smallest optogenetic tools for controlling PPIs and enable either green-ON/red-OFF (BICYCL-Red) or red-ON/green-OFF (BICYCL-Green) control with up to 800-fold state selectivity. The access to green wavelengths creates new opportunities for multiplexing with existing tools. We demonstrate the utility of BICYCLs for controlling protein subcellular localization and transcriptional processes in mammalian cells and for multiplexing with existing blue-light tools.

Abstract: Cyanobacteriochromes (CBCRs) are photoreceptors consisting of single or tandem GAF (cGMP-phosphodiesterase/adenylate cyclase/FhlA) domains that bind bilin chromophores. Canonical red/green CBCR GAF domains are a well-characterized subgroup of the expanded red/green CBCR GAF domain family that binds phycocyanobilin (PCB) and converts between a thermally stable red-absorbing Pr state and a green-absorbing Pg state. The rate of thermal reversion from Pg to Pr varies widely among canonical red/green CBCR GAF domains, with half-lives ranging from days to seconds. Since the thermal reversion rate is an important parameter for the application of CBCR GAF domains as optogenetic tools, the molecular factors controlling the thermal reversion rate are of particular interest. Here, we report that point mutations in a well-conserved W(S/G)GE motif alter reversion rates in canonical red/green CBCR GAF domains in a predictable manner. Specifically, S-to-G mutations enhance thermal reversion rates, while the reverse, G-to-S mutations slow thermal reversion. Despite the distance (>10 Å) of the mutation site from the chromophore, molecular dynamics simulations and nuclear magnetic resonance (NMR) analyses suggest that the presence of a glycine residue allows the formation of a water bridge that alters the conformational dynamics of chromophore-interacting residues, leading to enhanced Pg to Pr thermal reversion.

Abstract: Photoswitchable proteins enable specific molecular events occurring in complex biological settings to be probed in a rapid and reversible fashion. Recent progress in the development of photoswitchable proteins as components of optogenetic tools has been greatly facilitated by directed evolution approaches in vitro, in bacteria, or in yeast. We review these developments and suggest future directions for this rapidly advancing field.