Modular Protein-Pore Sensor for Transient PPI Detection
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Solution Overview
Problem
Current methods lack the capability to effectively and selectively detect transient protein-protein interactions at single-molecule resolution in a scalable and real-time manner, especially since proteins do not easily fit through nanopore sensors due to their size and require a transducing mechanism to convert physical associations into electrical signals.
Innovation Solution
A modular protein-pore based sensor is developed, featuring a membrane with a transducer, a protein receptor tethered by a flexible linker, and a charged polypeptide adaptor, using a monomeric β-barrel scaffold derived from FhuA, which allows for reversible current transitions across the membrane upon protein interaction, enabling selective and real-time detection of protein-protein interactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If proteins are detected using traditional nanopore sensors, then single-molecule detection capability is achieved, but proteins cannot effectively pass through the nanopore due to their size exceeding the pore diameter
Solution Approach 1:
The patent introduces a soluble adapter protein that acts as an intermediary between the target protein and the nanopore sensor. The adapter has a binding site that specifically captures the target protein in solution and presents it to the nanopore, enabling detection without the target protein needing to physically pass through the pore itself. This mediator resolves the size mismatch between large proteins and small nanopores.
2Loss of information
If physical associations of proteins are detected, then interaction information is obtained, but a transducing mechanism is required to convert reversible physical associations into electrical signals
Solution Approach 1:
The patent replaces complex mechanical transduction mechanisms with a simpler electrostatic sensing approach. The soluble adapter is designed with charged residues that create an electrostatic field detectable by the nanopore. When the target protein binds to the adapter, changes in the electrostatic field or ion current are detected, providing kinetic information about the interaction without requiring complex mechanical transducers.
3Productivity
If real-time detection of transient protein-protein interactions is achieved, then kinetic information is obtained, but detection sensitivity must be maintained in complex environments like fetal bovine serum
Solution Approach 1:
The patent applies local quality by designing the soluble adapter with specific local properties: charged residues positioned to create a distinctive electrostatic signature, and binding sites engineered for high specificity to the target protein. These localized features enable the adapter to maintain detection sensitivity in complex environments by creating a unique signal that can be distinguished from background noise in fetal bovine serum.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for the detection of protein-protein interactions at single-molecule resolution, providing kinetic information and distinguishing between binding and unbinding events, even in complex environments like fetal bovine serum, with high sensitivity and specificity.
Implementation Method 1
a transducing mechanism was required, converting the reversible physical associations and dissociations of the two protein partners in aqueous phase into a high-fidelity electrical signature of the nanopore sensor
Implementation Method 2
the addition of a protein target that interacts with the protein receptor into the cis side of the chamber will produce a reversible current transition across the membrane
Data Source
AI summary
A bioinspired protein pore-based nanostructure that can provide selective, real-time sampling of protein-protein interactions at single-molecule resolution. This modular nanostructure relied on a single polypeptide chain that encompassed a heavily truncated outer membrane protein, a highly flexible connector, a protein receptor element, as well as a polypeptide adapter. The presence of a protein ligand analyte in solution produced reversible binding and release events, in the form of discrete and stochastic current transitions between open substates of the transmembrane pore, the nature of which depend on both the amount of protein ligand analyte and the strength of the transient PPIs in aqueous phase.