Droplet Method for Bilayer Assays

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DESCRIPTION (provided by applicant): Membrane proteins are extremely important to understanding human health and treating disease. They make up 30% of all proteins in the body, are involved in regulating signal transduction and molecular transport into and between cells, and are the target of most drugs available today. To function properly, membrane proteins must be embedded in a lipid membrane layer. Well-controlled studies of individual membrane proteins require creating an artificial membrane to hold the protein, but existing methods for producing artificial membranes, such as the black lipid membrane and membranes with solid support, cannot be adapted to high- throughput techniques because the membranes are quite delicate for black lipid membranes or have limited access for membranes with solid support. High-throughput methods, which allow examination of a large number of compounds or a large number of interactions simultaneously, are increasingly being used to study molecular interactions affecting human health and to identify and test drugs. The importance of high- throughput screening has been recognized in the NIH Roadmap for Medical Research. The Molecular Libraries and Imaging portion of the NIH Roadmap seeks to facilitate the development of new, small-molecule drugs within the public sector. High-throughput screening of these small molecules is an integral part of the proposed effort. We propose to investigate a method for studying lipid membranes and membrane proteins based on an inverted emulsion. With this method we can readily produce bilayer membranes with small volumes and small surface areas. We expect that this method will be easily adapted to high-throughput screening of membrane protein function and drugs influencing membrane proteins. Because of the small size of the membrane, this technique will avoid the membrane fragility issues typical of black lipid membrane techniques. Furthermore, because the membrane is formed between two inverted emulsion droplets, we will avoid the difficulties in accessing both sides of the membrane found with artificial membranes on solid support. With the small volumes involved, we can minimize the quantities of reagent used in assays and can perform very large numbers of assays rapidly on a single substrate. We propose to investigate the inverted emulsion method for membrane proteins by: (1) studying bilayer formation in an inverted emulsions by characterizing the bilayer performance with respect to the continuous phase and lipid composition and phase of introduction, and comparing the membrane properties with liposomes through permeability studies; and (2) testing membrane performance using inserted proteins or peptides. We will incorporate one transmembrane protein and one self- assembled pore with a transmembrane peptide into the interfacial bilayer and use fluorescent dyes to establish their presence and function. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Transmembrane proteins are a very important class of molecules in the human body and are the target of most drugs available today. This research develops a powerful new method for studying the function of transmembrane proteins that should in turn lead to improvements in treating a wide range of diseases ranging from infections to cancer to genetic disorders.