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A pore-forming protein with a metal-actuated switch
B Walker1, J Kasianowicz, M Krishnasastry
1Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
Protein Engineering
|May 1, 1994
Summary
Researchers engineered a Staphylococcus aureus toxin to create a switchable pore. Introducing histidine residues allowed zinc ions to control pore activity, suggesting potential for metal ion sensors.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Staphylococcus aureus secretes alpha-hemolysin, a pore-forming exotoxin.
- This toxin forms hexameric pores in lipid bilayers, involving a central glycine-rich loop.
Purpose of the Study:
- To investigate the role of the central loop in alpha-hemolysin pore formation.
- To engineer a switchable pore by modifying the central loop.
Main Methods:
- Site-directed mutagenesis to replace amino acids 130-134 with five histidine residues.
- Planar lipid bilayer electrophysiology to record pore activity.
- Application of divalent zinc ions (Zn2+) and EDTA to modulate pore function.
Main Results:
- The histidine-substituted mutant exhibited switchable pore activity.
- Micromolar Zn2+ concentrations inhibited pore formation.
- EDTA reversed the inhibition, reactivating the pore.
- Zn2+ and EDTA acted on the pore from either side of the bilayer.
Conclusions:
- The central loop of alpha-hemolysin lines the conductive pathway and can be engineered for external control.
- Genetically engineered pore-forming proteins could serve as components in metal ion sensors.