Computational design of protein-based small-molecule biosensors

描述（由申请人提供）：检测信号并对其做出响应是生命系统最基本的能力之一。该应用旨在开发计算和实验设计策略，以设计新的基于蛋白质的传感器/致动器来响应细胞中的小分子。其关键思想是将小分子结合位点设计到异源二聚体蛋白质-蛋白质界面中，使得蛋白质-蛋白质相互作用变得依赖于小分子。两个蛋白质配偶体中的每一个将连接到分裂报告子的片段。然后功能传感器通过报告互补检测小分子的存在。以这种方式，传感器输出原则上是模块化的：不同的报告片段可以连接到小分子传感器组件，并测试检测（例如分裂GFP）或驱动（分裂酶或基因表达）。通过小分子诱导的二聚化来计算设计这种模块化传感器/致动器将是第一次。创建起始活性的中心策略是将配体蛋白单体结构的结合位点几何形状移植到蛋白质-蛋白质界面中，然后对界面进行计算重构。这种方法已经成功地设计了对焦磷酸法呢酯（FPP）（治疗分子、工业化学品和燃料合成途径中的中心中间体）做出反应的传感器，并为两个额外的目标产生了初始传感器活性。目标1旨在通过开发方法来解决计算设计的关键缺点，以（i）改善设计的结合位点几何形状;（ii）评估和限制结合位点残基的构象变异性;（ii）在DNA改组的计算等价物中重组来自设计集合的片段;以及（iv）改善设计的排名。目标2将建立一个实验平台，通过使用细胞中的模块化报告基因测试设计预测，筛选计算设计的文库，定向进化以优化传感器功能，以及体外生物物理表征来表征和改进工程传感器。这些研究将产生（i）可用于代谢工程应用的改进的FPP传感器和（ii）可用于特异性激活控制细胞信号传导的蛋白质-蛋白质相互作用的分子的新传感器，其建立在显示初始设计的传感器活性的初步数据的基础上。虽然小分子诱导的蛋白质二聚化存在于自然界中，并已被重新设计，但这些系统仅限于少数可以被检测到的分子，并且通常具有不希望的副作用。该项目中开发的方法可以大大拓宽小分子传感和驱动的应用。由于模块化方法允许表征许多功能和非功能设计，该项目还将提供关于设计的成功和局限性的独特信息，这对方法的改进至关重要。最终，这些研究将导致先进的计算设计方法，同时产生新的工具来控制生物工程应用中的细胞行为，并探索基础和疾病生物学。

DESCRIPTION (provided by applicant): Detecting signals and responding to them are among the most fundamental abilities of living systems. This application seeks to develop computational and experimental design strategies to engineer new protein-based sensor/actuators responding to small molecules in cells. The key idea is to engineer small-molecule binding sites into heterodimeric protein-protein interfaces such that the protein-protein interaction becomes dependent on the small molecule. Each of the two protein partners will be linked to a fragment of a split reporter. A functional sensor then detects the presence of the small molecule by reporter complementation. In this fashion, the sensor output is in principle modular: different reporter fragments can be attached to the small-molecule sensor components and tested for either detection (for example split-GFP) or actuation (split-enzymes or gene expression). Computational design of such modular sensor/actuators via small molecule-induced dimerization would be a first. The central strategy to create starting activity is to transplant binding site geometries from liganded protein monomer structures into protein-protein interfaces, followed by computational remodeling of the interface. This approach has led to successful design of sensors that respond to farnesyl pyrophosphate (FPP), a central intermediate in synthesis pathways for therapeutic molecules, industrial chemicals, and fuels, and generated initial sensor activity for two additional targets. Aim 1 seeks to address key shortcomings of computational design by developing methods to (i) improve designed binding site geometries; (ii) assess and restrict conformational variability of binding sites residues; (ii) recombine fragments from design ensembles in a computational equivalent of DNA shuffling; and (iv) improve ranking of designs. Aim 2 will build an experimental platform to characterize and improve engineered sensors by testing design predictions using modular reporters in cells, screening computationally designed libraries, directed evolution to optimize sensor function, and in vitro biophysical characterization. These studies will produce (i) improved FPP sensors useful in metabolic engineering applications and (ii) new sensors for molecules that could be used to specifically activate protein-protein interactions controlling cellular signaling, building on preliminary data showing initial designed sensor activity. While small molecule-induced protein dimerization exists in nature and has been reengineered, these systems are limited to a few molecules that can be sensed, and often have undesired side effects. The methodology developed in this project could greatly broaden applications of small-molecule sensing and actuation. Because the modular approach allows characterization of many functional and non- functional designs, this project will also provide unique information on successes and limitations of design that is critical for methodological improvements. Ultimately, these studies will lead to advanced computational design methods while generating new tools to control cellular behavior in biological engineering applications and to probe basic and disease biology.