Studies Toward A Self-Assembling Biosensor

Abstract

Almost all biological processes are driven by protein-protein interactions. It is therefore important to explore the underlying central principles that determine these interactions. Biosensors have been successfully used to investigate protein-protein interactions. The purpose of this research was to investigate the potential of a split-ion-channel system which is a novel concept, as a biosensor to study transient and rare protein-protein interactions that underpin all important biological processes. As an initial test system to this end, I have designed a model ion channel biosensor using a transmembrane peptide of the M2 influenza ion channel protein fused to a phosphorylation controlled coiled-coil partner using a spacer. The synthetic targets consisted of two different versions of the model system where one and two copies of the ion-channel fragment were covalently linked to the phosphorylation controlled coiled-coil via a spacer. Phosphorylation controlled folding of the coiled-coil was expected to re-constitute the split, and therefore potentially non-functional, ion-channel into a functional one. Both targets (one and two copies of M2TM attached to the coiled-coil) were successfully synthesized using Fmoc Solid Phase Peptide Synthesis and purified by RPHPLC. Detailed structural and functional analysis was carried out on the singly branched version in comparison to the control (ion-channel fragment by itself) using circular dichroism, colorimetric analysis and fluorescent dye efflux assays. Interesting observations were made which showed that the ion-channel fragment by itself was able to assemble into a functional entity both in the presence and absence of the coiled-coil partner, with only subtle differences observed in the fluorescent dye efflux assay. However, phosphorylation of the coiled-coil partner in the covalently linked system resulted in complete loss of secondary and tertiary structure resulting in loss of membrane disruption ability of the system as was observed in the colorimetric and fluorescent dye efflux assays. These results indicate that the M2TM fragment used in this study is not the ideal candidate as the reporter component of this model biosensor system. Further investigations using more accurate techniques such as electrical impedance spectroscopy might provide better insight into the observed marginal differences in the fluorescent dye efflux assay mentioned above and help to assess the appropriateness of the coiled-coil partner used in this study as the recognition element of the proposed biosensor system.