Using ion channel-forming peptides to quantify protein-ligand interactions.

Area of Science:

• Biophysics
• Biochemistry
• Analytical Chemistry

Background:

• Self-assembly of ion channel-forming peptides in lipid bilayers is crucial for membrane function.
• Sensing molecular interactions often requires sensitive and specific detection methods.

Purpose of the Study:

• To develop a method for sensing affinity interactions based on disrupting peptide self-assembly in lipid bilayers.
• To quantify monovalent ligand binding using ion channel conductance changes.

Main Methods:

• Utilizing ion channel-forming peptides (alamethicin derivative) in planar lipid bilayers.
• Covalently attaching a sulfonamide ligand to the peptide for binding carbonic anhydrase II (CA II).
• Measuring changes in ion channel conductance upon protein binding and release.

Main Results:

• Binding of carbonic anhydrase II (CA II) to the peptide-ligand conjugate inhibited ion channel conductance.
• Disruption of self-assembled pores was observed due to the interaction between CA II and the peptides.
• Addition of a competitive inhibitor restored current flow, allowing for quantification of binding.

Conclusions:

• The proposed method effectively senses affinity interactions by monitoring the disruption of ion channel self-assembly.
• This technique provides a quantitative measure of monovalent ligand binding, yielding a dissociation constant of approximately 2 microM for CA II.
• The results are consistent with independent measurements obtained via isothermal titration calorimetry.