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.
The immediate outcomes of this project will provide a fundamental and technological basis for accelerated discoveries in biosensor development. These proposed studies will impact healthcare by delivering the underlying rules and principles for the construction, composition, architecture, and function of selective nanodevices, which operate in complex biological fluids.
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