Development of Surface-Based Methods for Studies of Non-Electrolyte Membrane Transport

Licentiatavhandling, 2010

Membrane-transport proteins are a diverse family of proteins that all span the cell membrane where they control the transfer of nutrients, metabolites and ions into and out of the cell. Due to their vital role for the function of cells, reliable methods, preferably with high-throughput capacity, to study this class of proteins are essential in fundamental studies as well as in discovery and development of new drugs. Surface-based analytical techniques have been exploited extensively for analysis of biomolecular interactions in medical diagnostics and drug screening as well as in fundamental biochemical analysis. The combination of these techniques with platforms that mimics cell membrane, allows in vitro studies of membrane residing proteins. This thesis work deals with the use of surface-based methods for studying membrane transport processes, with a focus on transport of non electrolytes. A new self-assembly route implementing charged-stabilized bicelles and cholesterol-terminated DNA anchors was developed for the formation of multiple tethered lipid bilayers on solid supports with potential of being used in transport studies. Fluorescence imaging, quartz crystal micro balance with dissipation (QCM-D) and fluorescent recovery after photo-bleaching (FRAP) were used to monitor the formation and to characterize the multiple bilayers. Liposomes are cell like containers with the ability of hosting membrane proteins, which are used in functional studies a variety of membrane proteins including membrane protein transporters. A surface-based method, based on surface plasmon resonance (SPR) spectroscopy was explored for studying passive diffusion as well as membrane-protein mediated transport across lipid bilayers using surface-attached liposomes. The benefit of surface-based methods for this purpose is their potential to screen multiple reactions either sequentially or simultaneously, which we demonstrate in a functional study of the aquaglyceroporin, PfAQP, from the malaria parasite Plasmodium falciparum. Kinetics of transport of several sugar alcohols were successfully measured at different temperatures on the same set of reconstituted liposomes. The results proved the applicability of the method for membrane-protein mediated transport studies and shed light on the transport mechanisms of aquaglyceroporin. It turned out that the activation energies for facilitated diffusion of sugar alcohols with different length are the same, indicating that the rate of solute passage through the aquaglyceroporin pore is determined not by water-solute interactions, but by the frequency of successful collisions of sugar alcohol molecules with the pore entry of the channel. In addition, a careful investigation of measured kinetics revealed a heterogeneous nature of reconstituted liposomes with respect to the numbers of membrane proteins per liposome. To further investigate the encountered heterogeneity, a complementary strategy based on self-quenching of liposome-entrapped fluorescence dyes was defined, with the ultimate aim of studying fast (sub ms) transport events in single liposome level. Our preliminary results on performing such experiment on individual PfAQP reconstituted liposomes reflects the presence of two population of liposomes with either one or two membrane proteins, which is in good agreement with our SPR study.