Using ion channel-forming peptides to quantify protein-ligand interactions.
This paper proposes a method for sensing affinity interactions by triggering disruption of self-assembly of ion channel-forming peptides in planar lipid bilayers. It shows that the binding of a derivative of alamethicin carrying a covalently attached sulfonamide ligand to carbonic anhydrase II (CA II) resulted in the inhibition of ion channel conductance through the bilayer. We propose that the binding of the bulky CA II protein (MW approximately 30 kD) to the ion channel-forming peptides (MW approximately 2.5 kD) either reduced the tendency of these peptides to self-assemble into a pore or extracted them from the bilayer altogether. In both outcomes, the interactions between the protein and the ligand lead to a disruption of self-assembled pores. Addition of a competitive inhibitor, 4-carboxybenzenesulfonamide, to the solution released CA II from the alamethicin-sulfonamide conjugate and restored the current flow across the bilayer by allowing reassembly of the ion channels in the bilayer. Time-averaged recordings of the current over discrete time intervals made it possible to quantify this monovalent ligand binding interaction. This method gave a dissociation constant of approximately 2 microM for the binding of CA II to alamethicin-sulfonamide in the bilayer recording chamber: this value is consistent with a value obtained independently with CA II and a related sulfonamide derivative by isothermal titration calorimetry.
Mayer
Michael
M
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA. mimayer@umich.edu
Semetey
Vincent
V
Gitlin
Irina
I
Yang
Jerry
J
Whitesides
George M
GM
D000408:Alamethicin / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D002151:Calorimetry; D020128:Inhibitory Concentration 50; D007473:Ion Channels / Q000737:chemistry; D007477:Ions; D007700:Kinetics; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry; D015233:Models, Statistical; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D013449:Sulfonamides / Q000737:chemistry; D013997:Time Factors
D007473:Ion Channels; D007477:Ions; D008024:Ligands; D008051:Lipid Bilayers; D010455:Peptides; D011506:Proteins; D013449:Sulfonamides; D000408:Alamethicin