The proper function of integral membrane proteins is required for a number of essential cellular functions, and this class of proteins comprises the majority of drug targets. Despite the obvious significance of membrane proteins to matters of human health, our knowledge and understanding of their structure, function, and dynamics lags far behind that of soluble proteins. Although challenges are presented by overexpression, stabilization, purification, reconstitution and characterization, the growth of well-ordered single crystals of membrane proteins is still the most significant bottleneck for the structure determination of membrane proteins. The problems in the crystallization of membrane proteins are directly related to the fact that membrane proteins reside, and are stable and functional in a lipidic bilayer, whereas biochemical, molecular biological and biophysical studies and crystallization experiments are conducted in aqueous solution. Detergents have a complex behavior, forming numerous protein-detergent and detergent-detergent phases which are influenced by the physical chemical parameters used for crystallization such as the pH, ionic strength, viscosity, temperature etc. Furthermore, the structural and functional integrity of membrane proteins and their solubility strongly depend on the physical chemical properties of the phase behavior of the detergent. In this project, we propose a strategy for crystallization of membrane proteins that will eliminate the problems associated with protein-detergent complexes. We will develop a strategy to identify Rhodobacter membrane fractions that are enriched in overexpressed target membrane protein and will then incorporate these membranes directly into lipidic cubic materials for crystallization, circumventing the need for solubilization and purification steps that require detergents. The lipidic cubic matrices should provide the structural stabilization and dynamic flexibility necessary to conduct successful crystallization trials. Experiments will be carried out initially with a number of paradigmatic test membrane proteins, to be followed by the target proteins that will be expressed in Rhodobacter and provided by the Membrane Protein Production core facility.
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