Coupling a Live Cell Directed Evolution Assay with Coevolutionary Landscapes to Engineer an Improved Fluorescent Rhodopsin Chloride Sensor

Our understanding of chloride in biology has been accelerated through the application of fluorescent protein-based sensors in living cells. These sensors can be generated and diversified to have a range of properties using laboratory-guided evolution. Recently, we established that the fluorescent proton-pumping rhodopsin wtGR from Gloeobacter violaceus can be converted into a fluorescent sensor for chloride. To unlock this non-natural function, a single point mutation at the Schiff counterion position (D121V) was introduced into wtGR fused to cyan fluorescent protein (CFP) resulting in GR1-CFP. Here, we have integrated coevolutionary analysis with directed evolution to understand how the rhodopsin sequence space can be explored and engineered to improve this starting point. We first show how evolutionary couplings are predictive of functional sites in the rhodopsin family and how a fitness metric based on a sequence can be used to quantify the known proton-pumping activities of GR-CFP variants. Then, we couple this ability to predict potential functional outcomes with a screening and selection assay in live Escherichia coli to reduce the mutational search space of five residues along the proton-pumping pathway in GR1-CFP. This iterative selection process results in GR2-CFP with four additional mutations: E132K, A84K, T125C, and V245I. Finally, bulk and single fluorescence measurements in live E. coli reveal that GR2-CFP is a reversible, ratiometric fluorescent sensor for extracellular chloride with an improved dynamic range. We anticipate that our framework will be applicable to other systems, providing a more efficient methodology to engineer fluorescent protein-based sensors with desired properties.

我们对生物学中氯离子的认知，因活细胞内基于荧光蛋白的传感器（fluorescent protein-based sensors）的应用而得以加速推进。这类传感器可通过实验室指导的定向进化（directed evolution）进行构建与多样化改造，以获得一系列差异化特性。近期，我们证实了来自紫球蓝细菌（Gloeobacter violaceus）的野生型质子泵视紫红质wtGR，可被改造为氯离子荧光传感器。为解锁这一非天然功能，我们在与青色荧光蛋白（cyan fluorescent protein, CFP）融合的wtGR中，于希夫反离子（Schiff counterion）位点引入单个点突变D121V，由此获得GR1-CFP。本研究将共进化分析（coevolutionary analysis）与定向进化相结合，旨在探索并改造视紫红质的序列空间，以优化该起始改造靶点。我们首先证实，进化耦合可用于预测视紫红质家族的功能位点，同时基于序列的适应度指标可量化GR-CFP变体已知的质子泵活性。随后，我们将这一预测潜在功能结果的能力，与活大肠埃希菌（Escherichia coli）中的筛选与选择检测实验相结合，以缩小GR1-CFP质子泵通路上5个残基的突变搜索空间。这一迭代筛选流程最终获得GR2-CFP，其额外携带4个突变：E132K、A84K、T125C与V245I。最后，在活大肠埃希菌中开展的批量与单分子荧光测量结果显示，GR2-CFP是一种可逆的比率型胞外氯离子荧光传感器，且具备更优的动态范围。我们预计，本研究提出的研究框架可推广至其他系统，为按需改造具备目标特性的基于荧光蛋白的传感器提供更高效的技术路径。