In vitro studies of protein interactions on substrate supported artificial membranes

Abstract

Protein interactions that take place at the interface of a membrane are abundant within cellular organisms. Hence, surface coupled protein interactions are a matter of ongoing scientific investigation. In this work, elected protein interactions were studied at the interface of artificial membranes, attempting to create model systems that would best mimic the natural protein environment. In the first part of this work (chapter 4-6), a quartz crystal microbalance (QCM) biosen-sor assay based on solid supported membranes (SSMs) was established to quantify the inter-action of C-terminal polycystin-2 (cPC2) with its putative interaction partners C-terminal polycystin-1 (cPC1) and PIGEA14. It was found that the affinity of cPC2 to cPC1 was three times higher in the presence of Ca2+, leading to the assumption that cPC2 forms trimers in the absence of Ca2+. Based on the observation that the kinetic rate constants are Ca2+ dependent, a binding model was developed that describes a three step cPC2 binding in the absence and a one step binding in the presence of Ca2+. Following a hypothesis that had been suggested earlier, the interaction of cPC2 with PIGEA14 was investigated as a function of cPC2 pseudophosphorylation at Ser812. It was found that the affinity of the pseudophosphorylated mutant cPC2S812D to PI-GEA14 was indeed reduced two fold compared to cPC2wt. The second part of the thesis (chapter 7 and 8) dealt with investigating the interaction of filamentous actin (F-actin) with SSMs and pore spanning membranes (PSMs) on po-rous aluminum or silicon nitride based surfaces by means of optical waveguide spec-troscopy (OWS) and confocal laser scanning microscopy (CLSM). Mimicking the struc-ture of cellular microvilli, specific F-actin adsorption within and atop porous anodic aluminum oxide (AAO) films could be controlled using different functionalization strategies. The impact of a membrane bound F-actin network on the tension and viscoelastic properties of PSMs was investigated by means of atomic force microscopy (AFM). While the membrane tension remained almost unaltered, the origination of a viscoelastic membrane properties was caused by the attached F-actin network.