On-chip crystallization and in situ X-ray analysis of membrane proteins Summary Membrane Proteins play an important role in many biological processes as mediators of material and information across cellular and intracellular boundaries. Many diseases have been connected to the malfunction of membrane proteins but rational design of medical treatments can only occur once the 3D structure of a protein is known. Despite their role in many biological processes, and thus many diseases, the structural characterization of membrane proteins (MPs) has lagged significantly behind those of soluble proteins. The amphiphilic nature of MPs complicates growth of X-ray quality crystals for structural analysis. This project will advance microfluidic platforms for the in-meso crystallization of MPs. These platforms will be applied to resolve the structure and function of members of the heme-copper oxidase superfamily. The oxygen reducing members of this family are critical in cellular respiration, an essential biological function. Their malfunction can result in insufficient cellular energy production, which has been linked to several human genetic diseases. The promising in-meso approach prevents MPs from loosing their native conformation by maintaining them in an artificial membrane-like environment comprised of lipidic mesophases, from which crystals can be grown directly. We will develop microfluidic chips enabling nanoliter scale in-meso MP crystallization as well as subsequent on-chip, in situ X-ray analysis of MP crystals formed. In addition to reducing the amount of MP sample needed to <5 nL per test, this microfluidic approach will also eliminate direct handling of the often highly sensitive MP crystals between crystallization and X-ray analysis. In parallel, we will develop microfluidic chips for rapid screening of the phase behavior of lipid/water systems for their suitability for in-meso crystallization. A larger number of lipids for in-meso crystallization of MPs will provide a wider parameter space that can be screened for suitable crystal nucleation and growth conditions. Both, related efforts are expected to enhance the rate of membrane protein structure determination. Specific Aim 1: Develop and apply integrated microfluidic chips for in-meso crystallization screening and in situ X-ray structure determination of heme-copper respiratory oxidases. The proposed multi- compartment crystallization platforms will enable (i) screening for suitable crystallization conditions using <5 nL of MP solution per test, (ii) on-chip crystal quality screening via in situ X-ray analysis, as well as (iii) on-chip acquisition of high resolution data for MP structure determination of the most promising crystals under cryogenic conditions, all without off-chip handling of the often sensitive MP crystals. Specific Aim 2: Develop and apply integrated microfluidic chips for high throughput determination (via X-ray diffraction) of the phase behavior of lipids intended for the in-meso crystallization of MPs. The proposed multi-compartment platforms will be capable of formulating a range of different mesophase compositions for X-ray analysis over a range of temperatures. These chips will also allow for rapid study of the effects of various contaminants, such as small amounts of the detergents typically used in membrane protein isolation, on lipid/water phase behavior. PUBLIC HEALTH RELEVANCE: This project contributes to the study of membrane protein structure and indirectly to the elucidation of their function. Heme-copper oxidases have been linked to cellular respiratory diseases and are common drug targets. Improving our understanding of these proteins has the potential to advance medical treatment of the related genetically determined diseases in humans.