A functional protein pore with a "retro" transmembrane domain.

Area of Science:

• Protein engineering
• Biophysics
• Structural biology

Background:

• Functional proteins typically use unidirectional amino acid sequences.
• Retro sequences (reversed peptides) have not been incorporated into functional proteins.
• Beta-barrel proteins, like alpha-hemolysin, form pores through cell membranes.

Purpose of the Study:

• To investigate the feasibility of constructing functional protein structures using retro sequences.
• To characterize the properties of a beta-barrel pore formed by retrosequences.
• To explore the potential of beta-barrels to accommodate retro domains.

Main Methods:

• Engineering of retrosequences to form the transmembrane beta-barrel of alpha-hemolysin.
• Characterization of wild-type and retro heptamers using planar bilayer electrophysiology.
• Analysis of current-voltage relationships and single-channel conductance.
• Molecular modeling to understand structural implications.

Main Results:

• The entire transmembrane beta-barrel of alpha-hemolysin was successfully formed by retrosequences.
• Retro and wild-type heptamers exhibited similar properties in planar bilayers.
• Retro pores showed slightly reduced conductance and ohmic behavior compared to wild-type.
• Monomeric retro subunits showed premature assembly, but heteromeric pores could be formed.

Conclusions:

• Beta-barrels are highly adaptable to retrosequences, suggesting this fold may be uniquely suited for such constructs.
• The ability to form retro domains could explain the activity of membrane-permeabilizing peptides.
• Understanding the plasticity of the alpha-hemolysin beta-barrel aids in de novo design of membrane proteins.