Lu Xiaocen, Wen Yurong, Zhang Shuce, Zhang Wei, Chen Yilun, Shen Yi, Lemieux M Joanne, Campbell Robert E
Department of Chemistry, University of Alberta Edmonton Alberta T6G 2G2 Canada
Department of Biochemistry, University of Alberta Edmonton Alberta T6G 2H7 Canada.
Chem Sci. 2021 May 31;12(28):9658-9672. doi: 10.1039/d1sc01059j. eCollection 2021 Jul 21.
Photocleavable molecules can enable the light-dependent modulation of biomolecular activities with high spatiotemporal precision. We have previously reported a photocleavable protein (PhoCl1) that, uniquely, is a fully genetically encoded photocleavable molecule that can be introduced into cells in the form of its corresponding gene to enable optogenetic control of biomolecular activities. However, the first generation PhoCl1 exhibited a relatively slow rate of dissociation, potentially limiting its utility. Here, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Molecular dynamics (MD) simulations were performed to provide insight into the precise dissociation mechanism. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast ratio and PhoCl2f with faster dissociation. We characterized the performance of these new variants as purified proteins and in cultured cells. Our results demonstrate that PhoCl2 variants exhibit faster and more efficient dissociation, which should enable improved optogenetic manipulations of protein localization and protein-protein interactions in living cells.
光可裂解分子能够以高时空精度实现对生物分子活性的光依赖性调控。我们之前报道了一种光可裂解蛋白（PhoCl1），其独特之处在于它是一种完全通过基因编码的光可裂解分子，可以以其相应基因的形式导入细胞，从而实现对生物分子活性的光遗传学控制。然而，第一代PhoCl1表现出相对较慢的解离速率，这可能限制了它的实用性。在此，我们报道了PhoCl1绿色状态、红色状态和裂解后的空桶状结构的X射线晶体结构。进行了分子动力学（MD）模拟，以深入了解精确的解离机制。通过结构导向工程和定向进化，我们开发出了具有更高对比度的PhoCl2c和具有更快解离速度的PhoCl2f。我们对这些新变体作为纯化蛋白以及在培养细胞中的性能进行了表征。我们的结果表明，PhoCl2变体表现出更快、更有效的解离，这应该能够改善对活细胞中蛋白质定位和蛋白质-蛋白质相互作用的光遗传学操作。
