# Engineered Nanopores for Single-Molecule Stochastic Sensing

> **NIH NIH R01** · SYRACUSE UNIVERSITY · 2020 · $297,635

## Abstract

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.

## Key facts

- **NIH application ID:** 9987658
- **Project number:** 5R01GM088403-11
- **Recipient organization:** SYRACUSE UNIVERSITY
- **Principal Investigator:** LIVIU MOVILEANU
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $297,635
- **Award type:** 5
- **Project period:** 2009-09-28 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9987658

## Citation

> US National Institutes of Health, RePORTER application 9987658, Engineered Nanopores for Single-Molecule Stochastic Sensing (5R01GM088403-11). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9987658. Licensed CC0.

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