Solid-State NMR Structural Determination of Components in an Ion Channel Switch Biosensor

Abstract

High-resolution structural analysis of membrane proteins is difficult to achieve with the commonly used methods of structural biology, X-ray diffraction, and solution-state NMR spectroscopy. By combining solid-state NMR studies of membrane peptides in powder and oriented samples, with solution-state studies in detergent micelles, three dimensional molecular structures can be obtained. We have used solid-state NMR methods to study the antibiotic, gramicidin A (gA), a peptide that forms ion channels in bilayer lipid membranes (BLMs). Receptor groups have been linked to gA channels embedded in a BLM tethered to a gold electrode in an 'ion channel switch' biosensor. The receptor is attached to gA via a protein (either streptavidin or avidin) bonded to a biotin (B) moiety, with B in turn being covalently linked to the C-terminus of gA by one or more aminocaproyl (X) groups. Using NMR, we determined that the structures of these biotinylated gA analogues are very similar to native gA, and that biotinylated gA analogues with longer linkers (more X groups) are more accessible for binding to streptavidin/avidin and hence function better as ligand-gated ion channels.