Stability of functionalised supported lipid bilayers at ambient and physiological temperatures

Abstract

Supported lipid bilayers (SLBs) are functionalised with pMHC molecules, that are bound through His-tags to Ni-chelated lipids to study the activation behaviour of T cells. Consequently, stable protein binding and bilayer quality are important over long periods of time during experiments. In this thesis, the binding stability of His-tagged proteins has been determined in dependence on the number of tags at two temperature conditions. SLBs (98% POPC/2% DGS-NTA(Ni)) were functionalised with IE^K (two Histags) and Streptavidin (one/two/three His-tags). The stability of binding was observed at 21°C and 37°C with TIRF microscopy and protein surface densities were calculated over time. A desorption model was developed and off-rates were determined. At 21°C proteins stayed mobile on bilayers, although a decrease in density could be observed. With increasing number of His-tags binding was more stable and resulted in smaller off-rates. At 37°C proteins rapidly dissociated and only immobile aggregations were present within 2 − 2.5 hours. Off-rates increased and were in the same order of magnitude for each protein. Nevertheless, an improvement in binding stability could be achieved by increasing the pH of the buffer. Much slower desorption of IE^K was observed at 37°C. In conclusion, protein surface densities decreased both at 21°C and 37°C. Since the pH of the buffer may interfere with the coordination bond between His-tags and Ni-ions, experimental conditions must be chosen carefully.