Engineered Nanopores for Single-Molecule Stochastic Sensing

项目总结 对人类基因组的详细了解为许多物种的鉴定开辟了新的领域 与其他蛋白质有短暂的物理联系的功能性蛋白质。地球上的主要扰动 这些蛋白质-蛋白质相互作用(PPI)的强度导致疾病状况。然而，暂时性的本质是 在使用现有方法的异质解决方案中，很难对这些相互作用进行定量评估。这些 拟议的研究旨在通过创造基于孔的选择性传感器来应对这一挑战，该传感器可以检测 这些可逆的PPI在单绑定事件解析。精确的膜蛋白设计已经被用于 产生一个大电导的b桶跨膜孔，它可以容忍水溶蛋白的融合。 受体的整体结构不会恶化。当存在于溶液中的蛋白质配体与 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跃迁 在传感器的两个开放的子状态之间。这些毛孔管腔外的操作还没有 之前对其他跨膜蛋白进行了研究。许多这样的基于孔隙的传感器将被 被设计成单链多肽链蛋白，用于在正常和致癌条件下检测PPI。在这 项目中，这些含有受体的传感器将通过检测人类RAS的瞬时PPI而受到挑战 GTP酶与各种相互作用的伙伴，如效应器和其他调节蛋白。初级阶段 这一选择的动机是这种小GTP酶在细胞信号和癌症发展中的关键作用。这个 这些拟议研究的预期直接结果如下：(1)多路检测 可逆PPI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 相同的蛋白质受体；(Ii)构建、组成、结构、 以及包含人Ras GTP酶或Raf Ras结合的基于蛋白质孔的传感器的功能 结构域(Raf RBD)效应器，一种丝氨酸/苏氨酸特异的蛋白激酶；(Iii)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 (4)确定和量化致癌的RAS-RAF相互作用 哺乳动物细胞的清洁溶液和裂解物中的样品。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，或可能被进一步开发以创建高通量生物标记物的工具 血液、活组织检查和细胞裂解产物中的筛选和蛋白质图谱。

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.