Innovative Methods for Membrane Protein Crystalliza(RMI)

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DESCRIPTION (provided by applicant): This proposal focuses on the creation and validation of novel crystallization platforms and methodology to substantially speed up the process of determining optimal crystallization conditions for membrane proteins. The crystallization platform will enable precise control of the path towards supersaturation, to identify crystal-producing conditions for different protein samples. In preliminary studies, we have demonstrated the principle with soluble proteins. We have developed a microscale vapor-diffusion-based device that, by precise control of the rate of evaporation and by continuous monitoring, leads to the rapid identification of crystal producing conditions. Once such conditions have been identified, the procedure is repeated for further optimization to improve crystal quality and growth. The crystallization platform offers advances over current devices used for screening crystallization conditions. (1) Control over the rate of solvent evaporation allows one to adjust the kinetics of attaining supersaturation and, therefore, crystallization events. (2) Setup of initial crystallization conditions in multiple wells will be formulated automatically using integrated large-scale microfluidic networks. (3) Through the use of microfluidics, precipitins and other reagents such as detergents can be added to each crystallization well during the course of the experiment at any time, adding a new dimension for exploration of crystallization events. To this end microfluidic technology will be used, which will also reduce the amount of protein needed. We hypothesize that use of the advanced controls integrated within the proposed crystallization platforms will increase the success rate of identifying crystallization condition for membrane proteins. Specific Aim 1: Demonstrate the utility of the current crystallization platform for membrane protein crystallization. We will validate our existing vapor-diffusion-evaporation platform for the identification of crystal-producing conditions for known and novel membrane proteins. Specific Aim 2: Design, fabricate and validate new and innovative microscale crystallization platforms for membrane protein crystallization. Aims include the complete control over evaporation rate, the automatic loading, the automatic creation of composition gradients across different compartments, as well as subsequent addition of precipitins and other reagents, as required for a high throughput screening platform for membrane protein crystallization. Specific Aim 3: Experimental study and model development to increase understanding of phase behavior of solutions used for membrane protein crystallization. Light and X-ray scattering studies will be performed to unravel the phase behavior; in particular the kinetics involved in phase changes, of lipid - protein solutions as typically used in membrane protein crystallization. We also intend to extend our recently developed population-balance based model capable of predicting kinetic parameters nucleation and growth of soluble proteins.