Shedding Light: A Modified Phosphatidylinositol for the Investigation of Peptide Assemblies

Abstract

Phosphatidylinositolphosphates (from here on referred as PIPs) are phospholipids, composed of a polar inositol headgroup connected to a diacylglycerol (DAG) via a phosphate ester. Playing a crucial role in signal transduction pathways, PIPs mainly serve the purpose of enabling Ca2+-flux from the endoplasmatic reticulum, through enzymatic liberation of inositoltriphosphate (IP3). In recent years, work by Sitte and Schütz et al1,2 has shown that, apart from serving as a source for the second messenger IP3, PIPs are a critical factor in the stabilization of the serotonin transporter´s (SERT) oligomeric structure. In Sitte´s seminal work, strong evidence was found that PIPs ensure the structural integrity of SERT on the cell membrane through interaction of the polar inositol headgroup with basic patches, found on the surface of the transporter monomers. The assumption was further cemented by Schütz through implementation of a high-resolution single molecule microscopy technique (“Thinning Out Clusters While Conserving Stoichiometry of Labbeling”, TOCCSL3) developed in the former´s lab. To enable in-deep elucidation of the interactions between PIP and SERT, the need for a fluorescent and highly stable analogue of PIP has arisen. Therefore, the presented work will deal with the synthesis of such analogues, spanning from a simple model compound, as a proof-of-concept regarding the feasibility of chemical synthesis of such scaffolds, to structurally and functionally more complex and diverse members of the PIP-family. The structural modifications to the PIP-framework lead to photo-linkable and fluorescent representatives of this class of natural compounds. This will be achieved through introduction of handles enabling “click-chemistry” (e.g. azides) and thereby binding to fluorophores, as well as carbene donors (e.g. diazirines) for means of irreversible photo-linking to SERT on the site of the DAG.