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Engineering Enhanced Pore Sizes Using FhuA Δ1-160 from E. coli Outer Membrane as Template
Zhanzhi Liu1, Ishan Ghai2, Mathias Winterhalter2
1Institute of Biotechnology, RWTH Aachen University , Worringer Weg 3, 52074, Aachen, Germany.
ACS Sensors
|October 21, 2017
Summary
Researchers engineered a membrane channel, FhuA Δ1-160, to filter larger molecules by increasing beta-strands. This expanded the pore size up to 2.7 nm but faced stability limitations.
Area of Science:
- Biophysics
- Membrane protein engineering
- Molecular transport
Background:
- Biological membranes utilize channel proteins to regulate the passage of small molecules.
- The FhuA Δ1-160 mutant channel, lacking its cork domain, served as a starting point for pore expansion.
Purpose of the Study:
- To enhance the pore diameter of the FhuA Δ1-160 channel for filtering larger hydrophilic molecules.
- To investigate the relationship between increased beta-strand content and channel pore size and stability.
Main Methods:
- Stepwise insertion of beta-strand sequences into the FhuA Δ1-160 channel.
- Characterization of pore size using single-channel conductance measurements.
- Polymer exclusion assays with polyethylene glycol of varying sizes.
Main Results:
- Increasing beta-strands expanded the pore radius from 1.6 nm to a maximum of approximately 2.7 nm.
- Additional beta-strands increased ion current flickering and reduced channel stability.
- Excessive beta-strand integration led to decreased effective pore radii, indicating limitations.
Conclusions:
- The engineered FhuA channel variants show potential for filtering larger molecules.
- Channel stability and pore size are sensitive to the number of integrated beta-strands.
- This approach has limitations for achieving significantly larger, stable pores.
