Project Summary/Abstract Cell membranes are populated with proteins whose interactions are important for myriad cellular functions, from metabolism to signaling, including many functions implicated in processes such as neurodegeneration. Membrane proteins are notoriously difficult to handle and study. For many membrane proteins it is unknown whether they exist singly (as monomers), in pairs (as dimers), or larger collections (higher oligomers). A method to determine the number and arrangement of subunits in a protein complex within its native lipid membrane environment would resolve a number existing controversies, and would eventually have a large impact human health. One approach to this problem would be to study protein interactions at the single molecule level, which can require expensive and complex instrumentation. Another approach would be to employ a relatively inexpensive, chemically self-assembled “molecular hand” to program the interactions between precisely controlled numbers and ratios of proteins (their “stoichiometry”). Taking the second approach, we propose to develop a general platform for studying protein-protein interactions in lipid membranes, the DNA origami ring-templated liposome. This platform will allow exquisite control and measurement of protein-protein interactions within a single lipid bilayer, and overcome the limitations of existing methods for differentiating monomers from dimers. A DNA origami ring, filled with a disc-shaped liposomal membrane, will be constructed with attachment points for individual proteins of interest. Spaced with nanometer-precision along the edge of the ring, these attachment points will be used to define the number and type of protein subunits that can enter the membrane. Programmed release of the proteins into the membrane will be achieved through the introduction of DNA signals, that break DNA linkers between the proteins and the edge of the DNA ring. In our first aim, we will prototype and troubleshoot the platform by studying the interactions of fluorescently labelled DNA test molecules in a number of control experiments. In a second aim, we will replace the DNA test molecules with proteins having a known interaction, and verify that the platform can be used to measure protein-protein interactions. In a final aim, we will focus on resolving a long-standing question regarding the dimerization of a SNARE complex protein Synaptobrevin 2, which is an important participant in the membrane fusion process required for the release of neurotransmitters.