Engineered Nanopores for Single-Molecule Stochastic Sensing

项目摘要 对人类基因组的详细了解为鉴定许多 与其他蛋白质有短暂物理联系的功能蛋白质。主要扰动 这些蛋白质-蛋白质相互作用（PPI）强度导致疾病状况。然而， 这些相互作用难以使用现有方法在异质溶液中定量评估。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解决这一挑战， 这些可逆的PPI在单结合事件分辨率。精确的膜蛋白设计已被用于 产生一个大电导的b-桶跨膜孔， 受体而不破坏其整体结构。当存在于溶液中的蛋白质配体结合到 受体，配体的瞬时捕获和释放事件可以容易地记录为电流跃迁 在传感器的两个打开子状态之间。孔腔外的这些操作尚未被 以前在其他跨膜蛋白上进行。许多这样的基于孔的传感器将被 作为一个单一的多肽链蛋白质工程检查PPI在正常和致癌条件。在这 项目，这些含有受体的传感器将通过检查人类Ras的瞬时PPI来挑战 与各种相互作用的伙伴，如效应和其他调节蛋白的GT3。主 这种选择的动机是这种小GTdR在细胞信号传导和癌症发展中的关键作用。的 这些拟议研究的预期直接结果如下：（i）多重检测 可逆PPI，其使用对所述蛋白质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， 相同的蛋白质受体;（ii）开发用于构建，组成，结构， 和基于蛋白质孔的传感器的功能，所述传感器含有人Ras GT3或Raf Ras结合 结构域（Raf RBD）效应子，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）和交替框架折叠 (AFF)开关机制;（iv）使用 清洁溶液和哺乳动物细胞裂解物中的样品。这些选择性的传感器可以代表 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的工具 在血液、活组织检查和细胞裂解物中进行筛选和蛋白质分析。

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.