Engineered FhuA Protein Pore for Large Biomolecule Sensing
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Solution Overview
Problem
Current protein nanopores are limited by small diameter, restricting the sensing of large biomolecules and are multimeric in nature, making their engineering challenging due to numerous permutations and combinations of subunits.
Innovation Solution
Engineering a monomeric, β-barrel protein pore based on the bacterial outer-membrane protein FhuA from Escherichia coli, specifically removing the cork domain and modifying extracellular loops to create a wider pore with a measured conductance of approximately 3.9 nS, suitable for sensing large biomolecules and biomolecular complexes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If protein nanopores are engineered with larger diameter to detect large biomolecules, then the sensing capability for large biomolecules is improved, but the structural stability and functionality under various environmental conditions deteriorate
Solution Approach 1:
The invention divides the protein nanopore into distinct functional segments: a stable β-barrel core structure that maintains structural integrity, and engineered loops/domains that provide environmental stability. This segmentation allows the pore diameter to be enlarged while maintaining the stabilizing core structure.
Solution Approach 2:
The invention modifies specific parameters of the protein structure including enlarging the pore diameter while simultaneously adjusting amino acid sequences in loops and domains to enhance environmental stability. This parameter optimization resolves the contradiction between size and stability.
2Adaptability or versatility
If multimeric protein structures are used to achieve desired pore properties, then the functional versatility is improved, but the engineering complexity and design difficulty increase due to numerous permutations and combinations of subunits
Solution Approach 1:
The invention extracts and removes the multimeric assembly requirement from the design, focusing instead on engineering a stable, functional monomeric protein nanopore. This eliminates the complexity of dealing with multiple subunit permutations while retaining essential pore functions.
Solution Approach 2:
The monomeric protein nanopore is designed with universal functional capabilities through strategic placement of functional groups and engineered domains within the single subunit structure, achieving versatility without requiring multiple different subunits.
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
The engineered FhuA protein pore forms stable, large conductance channels capable of translocating bulky biopolymers, offering a versatile platform for biosensing and potential applications in medical biotechnology, with enhanced stability and functionality across various conditions.
Implementation Method 1
The resultant channel-forming protein possessed a measured conductance of ∼3.9 nS
Data Source
AI summary
A mutant Escherichia coli β-barrel monomeric protein pore bioengineered to remove a lumen-occluding domain and modify some of its extracellular domains. The modified protein, FhuA Δ/CΔ4L, forms a highly conductive pore as compared to other known pores and is capable of sensing large polypeptides and discriminating between modified protein analytes.