Engineered Nanopores for Single-Molecule Stochastic Sensing

用于单分子随机传感的工程纳米孔

• 批准号: 10227053
• 负责人: LIVIU MOVILEANU
• 金额: $ 29.74万
• 依托单位: SYRACUSE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-28 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227053
• 关键词: Address; Affinity; Apoptosis; Architecture; Bacillus amyloliquefaciens ribonuclease; Binding; Binding Sites; Biological; Biological Markers; Biopsy; Biosensor; Blood; Ca(2+)-Calmodulin Dependent Protein Kinase; Cells; Clinical; Complex; Coupled; Detection; Deterioration; Development; Disease; Electrostatics; Elements; Engineering; Entropy; Equilibrium; Equipment; Event; GTP Binding; Genetically Engineered Mouse; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Healthcare; Human; Human Genome; Integral Membrane Protein; Ions; Kinetics; Knowledge; Laboratories; Lead; Ligands; Liquid substance; Malignant Neoplasms; Mammalian Cell; Measurement; Membrane Proteins; Methods; Monomeric GTP-Binding Proteins; Motivation; Nature; Oncogenes; Oncogenic; Outcome; Phase; Phenotype; Physiologic pulse; Physiological; Pore Proteins; Process; Protein Engineering; Protein Kinase; Proteins; Protocols documentation; Ras/Raf; Reaction; Resolution; Ribonucleases; Role; Sampling; Serine; Signal Transduction; Specificity; Structure; System; Techniques; Technology; Thermodynamics; Threonine; Time; Water; aqueous; base; cell growth; cofactor; data acquisition; design; frontier; genetic regulatory protein; hydrophilicity; inhibitor/antagonist; interfacial; multiplex detection; nanodevice; nanopore; nanoproteomic; novel; personalized medicine; polypeptide; protein complex; protein function; protein profiling; protein protein interaction; ras GTPase-Activating Proteins; ras Proteins; receptor; response; screening; sensor; single molecule; therapeutic target; tool; tumorigenic

Project summary Detailed knowledge of the human genome has opened up a new frontier for the identification of many functional proteins involved in brief physical associations with other proteins. Major perturbations in the strength of these protein-protein interactions (PPI) lead to disease conditions. However, the transient nature of these interactions is difficult to assess quantitatively in heterogeneous solutions using existing methods. These proposed studies are aimed at addressing this challenge by creating selective pore-based sensors that detect these reversible PPI at single-binding event resolution. Precise membrane protein design has been used to produce a large-conductance b-barrel transmembrane pore that tolerates the fusion of a water-soluble protein receptor without deterioration of its overall structure. When a protein ligand present in solution binds to the receptor, transient capture and release events of the ligand can be readily recorded as current transitions between two open substates of the sensor. These manipulations outside the pore lumen have not been conducted previously on other transmembrane proteins. A number of such pore-based sensors will be engineered as a single-polypeptide chain protein to examine PPI in normal and oncogenic conditions. In this project, these receptor-containing sensors will be challenged by inspecting transient PPI of the human Ras GTPase with various interacting partners, such as effectors and other regulatory proteins. The primary motivation for this choice is the pivotal role of this small GTPase in cell signaling and cancer development. The expected immediate outcomes of these proposed studies will be the following: (i) the multiplexed detection of reversible PPI using composite mixtures of protein ligands of varying binding affinity and specificity for the same protein receptor; (ii) the development of rules and principles for the construction, composition, structure, and function of protein pore-based sensors that contain either a human Ras GTPase or a Raf Ras binding domain (Raf RBD) effector, a serine/threonine-specific protein kinase; (iii) the detection of PPI using guanine nucleotide exchange factors (GEFs), Ras GTPase activating proteins (GAPs), and alternate frame folding (AFF) switch mechanisms; (iv) the identification and quantification of oncogenic Ras-Raf interactions using samples in clean solutions and lysates of mammalian cells. These selective sensors could represent the basis for a nanoproteomics platform or might be further developed to create tools for high-throughput biomarker screening and protein profiling in blood, biopsies, and cell lysates.

项目概要 人类基因组的详细知识为识别许多基因开辟了新领域 与其他蛋白质有短暂物理联系的功能性蛋白质。主要扰动 这些蛋白质-蛋白质相互作用（PPI）的强度会导致疾病。然而，瞬态性质 使用现有方法很难在异构解决方案中定量评估这些相互作用。这些 拟议的研究旨在通过创建选择性的基于孔隙的传感器来应对这一挑战 这些可逆的 PPI 在单结合事件分辨率下。精确的膜蛋白设计已被用于 产生大电导 b 桶跨膜孔，耐受水溶性蛋白质的融合 受体而不破坏其整体结构。当溶液中存在的蛋白质配体与 受体、配体的瞬时捕获和释放事件可以很容易地记录为电流转换 传感器的两个开放子状态之间。这些孔腔外的操作尚未被 之前对其他跨膜蛋白进行过。许多这样的基于孔隙的传感器将被 被设计为单多肽链蛋白，用于在正常和致癌条件下检查 PPI。在这个 项目中，这些含有受体的传感器将面临检查人类 Ras 瞬时 PPI 的挑战 GTPase 与各种相互作用的伙伴，例如效应子和其他调节蛋白。初级 这一选择的动机是这种小 GTP 酶在细胞信号传导和癌症发展中的关键作用。这 这些拟议研究的预期直接结果如下：（i）多重检测 使用具有不同结合亲和力和特异性的蛋白质配体的复合混合物的可逆 PPI 相同的蛋白质受体； (ii) 制定建造、组成、结构的规则和原则， 含有人 Ras GTPase 或 Raf Ras 结合的基于蛋白质孔的传感器的功能和功能 结构域 (Raf RBD) 效应子，一种丝氨酸/苏氨酸特异性蛋白激酶； (iii) 使用鸟嘌呤检测 PPI 核苷酸交换因子 (GEF)、Ras GTP 酶激活蛋白 (GAP) 和交替框折叠 (AFF) 切换机制； (iv) 致癌 Ras-Raf 相互作用的鉴定和定量 清洁溶液中的样品和哺乳动物细胞裂解物。这些选择性传感器可以代表基础 用于纳米蛋白质组学平台或可能进一步开发以创建高通量生物标志物工具 血液、活检和细胞裂解物中的筛选和蛋白质分析。