The photocleavable protein (PhoCl) is a green-to-red photoconvertible fluorescent protein that, when illuminated with violet light, undergoes main chain cleavage followed by spontaneous dissociation of the resulting fragments. The first generation PhoCl (PhoCl1) exhibited a relative slow rate of dissociation, potentially limiting its utilities for optogenetic control of cell physiology. In this work, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. 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 expressed 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.

中文翻译：

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具有更快，更高效解离的改良光裂解蛋白

可光裂解的蛋白质（PhoCl）是一种绿色到红色的可光转换的荧光蛋白质，当用紫光照射时，会发生主链裂解，然后自发解离所得片段。第一代PhoCl（PhoCl1）表现出相对较慢的解离速率，可能会限制其用于光遗传学控制细胞生理学的效用。在这项工作中，我们报告了P​​hoCl1绿色状态，红色状态和裂开的空桶的X射线晶体结构。使用结构指导的工程技术和定向进化技术，我们开发了具有更高对比度的PhoCl2c和具有更快解离的PhoCl2f。我们表征了这些新变体作为纯化蛋白的性能，并在培养细胞中表达。我们的结果表明，PhoCl2变体显示出更快，更有效的解离，