Supported Cell Membrane Mimics - with Incorporated Transmembrane Proteins
Doctoral thesis, 2003
Supported lipid membranes provide the natural environment for cell membrane constituents such as transmembrane proteins. To allow the use of surface analytical tools for studies of transmembrane proteins, protocols that allow the protein to reside within the supported membrane in a native state need to be developed. In biosensing applications, including drug screening and medical diagnostics, as well as fundamental studies of transmembrane proteins, surface-based techniques have turned out to be important analytical tools, thus complementing the biochemistry and molecular biology methods, central to this field. Two different surface assemblies of lipids and proteins were prepared and investigated in this thesis work: supported planar proteolipid bilayers and supported intact proteoliposomes. A common method for preparation of supported planar proteolipid bilayers, used in this work, is the vesicle rupture technique where liposomes or proteoliposomes, consisting of phospholipids and transmembrane proteins, are exposed to a SiO2-surface were they rupture and thereby form a supported bilayer, which mimics many aspects of native cell membranes. The proteoliposome rupture and fusion process for preparation of supported proteolipid membranes was studied with the quartz crystal microbalance (QCM-D), surface plasmon resonance (SPR), and atomic force microscopy (AFM) techniques. The influence of different transmembrane proteins and different protein-concentrations on the bilayer formation process was investigated. It was found that transmembrane proteins, which do not have significant hydrophilic parts exposed outside the vesicle, have relatively minor effects on the bilayer formation process, compared to pure lipid vesicles. In contrast, when the protein contains hydrophilic parts, bilayer formation seems severely hampered. These results have important implications for the construction of stable cell membrane-mimicking assemblies utilizing transmembrane proteins in, for example, drug screening devices, as well as for fundamentals studies of cell membrane components. Based on the problems encountered using planar supported protein containing bilayers, a complementary strategy for supported membranes was also investigated. In this part, multilayers of proteoliposomes were immobilized on chemically modified Au or SiO2 surfaces. The multilayer vesicle assemblies were investigated using QCM-D and SPR. In particular, it was shown that at least six layers could be efficiently formed, thus allowing to fill the full sensing depth of QCM-D and SPR techniques. In this way, a signal-amplification for studies of membrane components was proven. It was also demonstrated that the multilayer assembly had, except for the bottom layer, a negligible influence on the activity of the proton pumping protein Transhydrogenase.