Computational design of protein-based small-molecule biosensors

基于蛋白质的小分子生物传感器的计算设计

• 批准号: 9274033
• 负责人: Tanja Kortemme
• 金额: $ 7.58万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9274033
• 关键词: Acids; Address; Adverse effects; Affinity; Algorithms; Behavior; Behavior Control; Binding; Binding Sites; Biological; Biological Process; Biology; Biomedical Research; Biophysics; Biosensor; Cell physiology; Cells; Chemicals; Complement; Computer software; Computing Methodologies; Coupled; Coupling; DHFR gene; DNA Shuffling; Data; Detection; Development; Dimerization; Directed Molecular Evolution; Disease; Engineering; Enzymes; Escherichia coli; Experimental Designs; Foundations; Funding; Gene Expression; Geometry; Health; Hydrogen Bonding; Ibuprofen; In Vitro; Knowledge; Lead; Learning; Letters; Libraries; Life; Ligands; Link; Metabolic; Methodology; Methods; Molecular; Nature; Organism; Output; Pathway interactions; Production; Protein Engineering; Protein Region; Proteins; Reporter; Research; Research Personnel; Robotics; Side; Signal Transduction; Signaling Protein; Sirolimus; Structure; System; Tacrolimus Binding Protein 1A; Technology; Testing; Therapeutic; Training; Transplantation; Vertebral column; Work; base; biophysical properties; design; farnesyl pyrophosphate; improved; improved functioning; in vivo; member; metabolic engineering; model design; monomer; new technology; practical application; programs; protein function; protein protein interaction; response; scaffold; screening; sensor; small molecule; success; tool; transcription factor

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.

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，以设计响应细胞中小分子的新型基于蛋白质的传感器/执行器。关键思想是将小分子结合位点设计到异二聚体蛋白质-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。两个蛋白质伴侣中的每一个都将与分裂报告基因的片段连接。然后，功能传感器通过报告基因互补来检测小分子的存在。通过这种方式，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上，并进行检测（例如分裂-GFP）或驱动（分裂酶或基因表达）测试。通过小分子诱导二聚化的这种模块化传感器/执行器的计算设计将是第一个。创建起始活性的核心策略是将结合位点几何形状从配体蛋白质单体结构移植到蛋白质-蛋白质界面中，然后对界面进行计算重构。这种方法成功地设计了对法尼基焦磷酸 (FPP) 做出反应的传感器，法尼基焦磷酸是治疗分子、工业化学品和燃料合成途径中的核心中间体，并为另外两个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主要缺点：(i) 改进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) 以相当于 DNA 改组的计算方式重组来自设计整体的片段； (iv) 提高设计的排名。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器测试设计预测、筛选计算设计的库、优化传感器功能的定向进化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，以及（ii）新的分子传感器，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，以显示初始设计的传感器活性的初步数据为基础。虽然小分子诱导的蛋白质二聚化存在于自然界中并且已被重新设计，但这些系统仅限于可感知的少数分子，并且通常会产生不良副作用。该项目开发的方法可以极大地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性和非功能性设计，因此该项目还将提供有关设计的成功和局限性的独特信息，这对于方法改进至关重要。最终，这些研究将带来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生物学和疾病生物学。