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.

项目摘要 对人类基因组的详细知识为识别许多人开辟了一个新的领域 功能蛋白与其他蛋白质的短暂物理关联有关。在 这些蛋白质蛋白相互作用（PPI）的强度导致疾病。但是，瞬态的性质 这些相互作用很难使用现有方法在异质溶液中进行定量评估。这些 提出的研究旨在通过创建基于选择性孔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用于 产生大传统的B桶跨膜孔，该孔可耐受水溶性蛋白的融合 受体而不会恶化其整体结构。当溶液中存在的蛋白质配体与 受体，瞬态捕获和配体的释放事件可以很容易地记录为当前过渡 在传感器的两个开放式取代之间。这些操纵孔管腔外没有 先前在其他跨膜蛋白上进行。许多这样的基于孔的传感器将是 设计为单型链肽链蛋白，以在正常和致癌条件下检查PPI。在这个 项目，通过检查人类RAS的瞬时PPI，这些含受体的传感器将受到质疑 具有各种相互作用伴侣的GTPase，例如效应子和其他调节蛋白。主要 这种选择的动机是这种小GTPase在细胞信号传导和癌症发展中的关键作用。这 这些提出的研究的预期直接结果将是以下内容：（i）多路复用的检测 可逆PPI使用不同结合亲和力的蛋白质配体的复合混合物和特异性的特异性 相同的蛋白质受体； （ii）制定建筑，组成，结构的规则和原则， 蛋白孔基的传感器的功能，该传感器包含人Ras GTPase或RAF RAS结合 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（GEFS），RAS GTPase激活蛋白（GAP）和替代框架折叠 （AFF）开关机制； （iv）使用使用的鉴定和定量使用 哺乳动物细胞的干净溶液和裂解物中的样品。这些选择性传感器可以代表基础 对于纳米蛋白质组学平台，或可能进一步开发以创建用于高通量生物标志物的工具 血液，活检和细胞裂解物的筛查和蛋白质分析。