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Porin mutants with new channel properties
B Schmid1, L Maveyraud, M Krömer
1Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Freiburg im Breisgau, Germany.
Protein Science : a Publication of the Protein Society
|July 31, 1998
Summary
Researchers engineered Rhodopseudomonas blastica diffusion porins by creating mutants. These modified porins exhibit altered diffusion properties and structures, demonstrating rational pore engineering is achievable.
Area of Science:
- Biochemistry
- Structural Biology
- Membrane Protein Engineering
Background:
- The general diffusion porin from Rhodopseudomonas blastica serves as a model for studying transport.
- Production of porins in Escherichia coli inclusion bodies allows for large-scale studies.
- Reconstitution into native structures is crucial for functional analysis.
Purpose of the Study:
- To engineer the diffusion porin from Rhodopseudomonas blastica.
- To investigate the impact of mutations on porin diffusion properties and structure.
- To explore the potential for rational pore engineering.
Main Methods:
- Production of Rhodopseudomonas blastica diffusion porin in Escherichia coli inclusion bodies.
- Refolding of porins to their native structure.
- Creation and characterization of 13 pore eyelet mutants.
- Planar lipid bilayer experiments to measure diffusion properties.
- X-ray crystallography to determine the structures of seven mutants.
Main Results:
- Mutations at the pore eyelet altered diffusion properties.
- Charge-modifying mutations confirmed cation selectivity.
- Deletions in loop L3 led to unstable trimers and pores.
- Reducing eyelet cross-section with tryptophan decreased conductivity.
- A mutant with six tryptophans exhibited voltage-dependent closure, suggesting two states.
Conclusions:
- The pore eyelet is a key site for engineering porin diffusion.
- Rational design can modify porin function and structure.
- Engineered porins show potential for controlled transport applications.