Biology subjects

Woolley, A.

Publications and source records attributed to Woolley, A..

4 recordsLinked to original sources

bioRxiv2025

An ultra-low background far-red light-responsive optogenetic tool based on an engineered biliverdin-binding domain

The robustness and broad applicability of an optogenetic tool depends heavily on the properties of the underlying photoreceptor protein and its cognate binding partner - the light responsive core. Current red light optogenetic systems for use in mammalian cells all rely on phytochrome based photoreceptors. These are large (70 kDa) proteins that act as dimers, thereby enforce dimerization on attached proteins. Naturally occurring or engineered binding partners can function effectively in certain cases, but large size, complex mode of interaction, background binding, relatively weak affinity and/or low fold changes between on and off states are significant limitations. Using structure-based design and directed evolution we developed a small (17 kDa) monomeric bilverdin binding photoreceptor FenixS, and a highly selective, high-affinity binder, Ash1 (6 kDa). Negligible off-state binding and a >1200-fold increase in binding affinity upon 700 nm illumination result in a high performance, ultra-low background, light responsive core for a diverse range of applications. An optogenetic tool for red light activation of transcription in mammalian cells based on the FenixS-Ash1 core exhibits robust performance without the need for biliverdin supplementation.

synthetic biology↗

bioRxiv2025

Red-light-only control of protein-protein interactions using a cyanobacteriochrome (UNICYCL)

Most optogenetic tools are controlled by blue light. Red-light responsive tools enable multiwavelength applications and allow greater biological tissue penetration with reduced toxicity. Current red-light tools are primarily based on phytochromes, large dimeric proteins with a structurally complex mode of interaction with their binding partners. Here we introduce a small red-light-only responsive system composed of a GA domain (6 kDa) that binds to a CBCR GAF domain (17 kDa) with a Kd [~]1-5 {micro}M to form a 1:1 complex in the dark. Red light causes dissociation of the complex by causing a >25-fold decrease in binding affinity. The CBCR GAF domain reverts to the dark state with a half-life of [~]1 min and the complex reforms. Structural analysis using NMR measurements combined with molecular docking and dynamics simulations shows that the binder interacts with the GAF domain and senses isomerization of the bilin chromophore at a site that overlaps the critical tongue domain of phytochromes. This system provides a small, simple red-light-only optogenetic tool that can operate to control protein-protein interactions in vitro and in living cells.

biochemistry↗

bioRxiv2020

Critical role for cold shock protein YB-1 in cytokinesis

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and cancer progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cell lines resulted in cytokinesis failure, multinucleation and an increase in G1 transit time. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging of cells undergoing cytokinesis both in vitro and in zebrafish embryos, we found that YB-1 was critical for microtubule organization during cytokinesis. Using mass spectrometry we identified multiple novel phosphorylation sites on YB-1. We show that phosphorylation of YB-1 at multiple serine residues was essential for its function during cytokinesis. Using atomistic modelling we show how multiple phosphorylations alter YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining for the first time precisely how YB-1 regulates cell division. SUMMARYY-box-binding protein-1, YB-1, is essential for cell division, but it is not clear how it functions. Using live imaging and confocal microscopy we show that YB-1 functions only in the last step of division, specifically being required to initiate cytokinesis.

cell biology↗

bioRxiv2019

Green, orange, red, and far-red optogenetic tools derived from cyanobacteriochromes

Existing optogenetic tools for controlling protein-protein interactions are available in a limited number of wavelengths thereby limiting opportunities for multiplexing. The cyanobacteriochrome (CBCR) family of photoreceptors responds to an extraordinary range of colors, but light-dependent binding partners for CBCR domains are not currently known. We used a phage-display based approach to develop small (~50-residue) monomeric binders selective for the green absorbing state (Pg), or for the red absorbing state (Pr) of the CBCR Am1_c0023g2 with a phycocyanobilin chromophore and also for the far-red absorbing state (Pfr) of Am1_c0023g2 with a biliverdin chromophore. These bind in a 1:1 mole ratio with KDs for the target state from 0.2 to 2 M and selectivities from 10 to 500-fold. We demonstrate green, orange, red, and far-red light-dependent control of protein-protein interactions in vitro and also in vivo where these multicolor optogenetic tools are used to control transcription in yeast.

synthetic biology↗