Unraveling the interaction networks among functional proteins is essential in fundamental and clinical biomedical diagnostics by providing a mechanistic understanding of the complex regulatory processes of the cell, identifying their relationships to diseases, accelerating protein biomarker discovery, and assisting drug design. Advances in rational membrane protein design, chemical modification, biomolecular recognition, and single-molecule science will be used in concert for the creation of a new methodology to sample protein-protein interactions at high temporal and spatial resolution, as well as for the detection, exploration, and characterization of individual proteins. These proposed studies are aimed at engineering protein nanopore- based sensing devices featured by ligand-containing flexible tethers. Ample redesign of ferric hydroxamate uptake component A (FhuA), a monomeric b-barrel protein with a remarkable array of advantageous traits, such as robustness, versatility, and tractability, will result in a unique nanostructure with a single tethered proteinor DNA aptamer ligand at a strategic positioning of the nanopore. The FhuA-based scaffold is an attractive choice for this task, because it's open-state, quiet current remains stable for long periods within an unusually broad range of detection circumstances. These benefits will be used in various biosensing schemes, in which individual protein-protein and protein-DNA recognition events will produce detectable, discrete and reversible changes in the conformational dynamics of the movable tether, inducing alterations in the single- channel electrical signature. The expected immediate outcomes will be the following: (i) the creation of sensing elements for examining protein-protein interactions under equilibrium and non-equilibrium conditions; (ii) the development of highly specific nanopore-based sensing elements for a protein biomarker; (iii) a better understanding of the impact of tunable and constraining tethers on the intermolecular forces among protein partners, which has implications for the in vivo contexts of complex recognition events produced by anchored protein domains; (iv) the improvement in the sensitivity of the single-molecule detection of protein-protein interfaces, pushing forward the nanopore technology for the disentanglement of weak protein-protein interactions; (v) the expansion of the modularity and scalability of engineered protein nanopores as well as their integration with a synthetic membrane, improving their mechanical, thermal, electrical, and chemical stability. The adaptation of these unusual nanostructures with movable arms to an integrated microfabricated chip platform will provide a new generation of research tools for exploring the molecular basis of protein-protein recognition events in a sensitive, specific and quantitative fashion.
Design and development of highly specific and sensitive protein-based detectors will accelerate discoveries in fundamental and clinical molecular biomedical diagnostics, with applications in several realms, including prognosis and detection of cancers at earlier stages than currently possible, as well as protein biomarker profiling. In response to these pressing needs, this grant application is aimed at expanding nanopore technology to that of real-time protein detection and analysis, which will represent a critical ste towards the creation of high-throughput devices for targeted protein sampling.
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