Membrane proteins are responsible for key cell signaling and material/energy transduction processes in biology. Many diseases have been connected to the malfunction of membrane proteins but the rational design of medical treatments can only occur once the 3D structure of a protein is known. However, the amphiphilic nature of membrane proteins complicates the growth of high quality crystals for structural analysis by X-ray diffraction. Moreover, their limited availability hampers high-throughput screening efforts to determine suitable crystallization conditions. Of the >47,000 structures deposited in the Protein Databank less than 300 are for membrane proteins, and of these only a tiny fraction are human membrane proteins, despite genomic estimates that -30% of proteins are expected to be integral membrane proteins. This disparity represents an area of critical need for the development of new methods for crystallization given the key role that membrane proteins play in pathways related to many diseases. I propose the elucidation of the structure and operational mechanism of certain members of a class of heme-copper oxidase membrane proteins. The oxygen reducing members of this family of proteins are critical enzymes in the respiration process of the cell. A number of human genetic diseases have been tied to oxidase malfunction, and respiratory oxidases have potential as drug targets for pathogenic bacteria. A microscale crystallization method will enable determination of suitable crystallization conditions while using miniscule amounts of protein (nL to pL instead of uL scale) and maintaining the protein in a membrane-like environment. Specific Aim 1. Finalize the design, fabrication, and validation of our microfluidic crystallization platforms that integrate fluid metering, mixing, and precipitant addition to screen for potential crystallization conditions by the in-meso method. In this method, the protein resides in a membrane-like, lipidic mesophase during crystallization, enhancing the chance of obtaining a high quality crystal. Specific Aim 2. Elucidate the structure and biological mechanisms of various bacterial members of the heme-copper respiratory oxidase superfamily of membrane proteins after using the microfluidic platforms of SA1 to identify in-meso crystallization conditions. More specifically, this method will be applied to (a) improve the structural information on proteins/mutants of which only low resolution structural information is available and (b) obtain the structure of novel protein constructs or mutants of which the structure has not previously been resolved. Structure-function studies will be performed in order to extend biological knowledge of the molecular mechanism behind their function. PUBLIC HEALTH RELEVANCE The relevance of this research to public health lies in the elucidation of the mechanism whereby various enzymes associated with cellular energy production function, thus facilitating and expediting the understanding of diseases associated with respiratory oxidase membrane proteins, and guiding the development of various pharmaceutical treatments.