In vitro Synthesis of GPCRs in a Solid Supported Planar Lipid Membrane System
Membrane protein research depends largely on the availability of functional protein in an instrumental set-up for application of biophysical investigations. We used an in vitro synthesis strategy in the presence of a solid supported lipid membrane as a new experimental strategy for the expression and membrane insertion of complex membrane proteins. Thus we realized the insertion of such a complex membrane protein, the odorant receptor OR5 from rat, which belongs to the highly important class of G-Protein coupled receptors (GPCRs). By applying a set of different biophysical and biochemical tools, e.g. surface plasmon enhanced fluorescence spectroscopy (SPFS), immunological detection (ELISA), control of integration based on enzymatic digestion, we proofed that membrane insertion of the protein is complete, proceeds in a vectorial manner and provides the odorant receptor in a biological active form, which recognizes the corresponding ligand molecule, the odour lilial. This experimental system of in vitro expressed and tBLM inserted OR5 was further used for single molecule measurements with image correlation microscopy, a combination of total internal reflection fluorescence (TIRF) and fluorescence correlation spectroscopy (FCS). The measurements focused on the visualization of odorant receptor molecules and clusters, respectively, as well as the determination of their lateral diffusion within the artificial membrane system. We show the fluorescently labeled antibodies tagging affinity labels of the individual OR5 proteins, inserted in the membrane system. High-resolution image correlation microscopy, i.e. the spatio-temporal auto-correlation of the membrane proteins, yields information about the spatial protein distribution (incorporation density) as well as the protein mobility (diffusion in the membrane).
Record created on 2009-10-27, modified on 2016-08-08