This award is for the development of a new instrument called the Phase Diagram Visualizer (PDV). The PDV will be used to optimize the conditions for crystallizing proteins. This has a large potential value, because the ability to crystallize proteins is currently a bottleneck in efforts to determine protein structures. The PDV is a novel microfluidic device for creating overlapping concentration gradients of protein, precipitant, and buffer. Crystal formation will be visualized, and software will determine the phase diagram of the system, based on the diffusion constants for the protein and other solutes. The PDV's microfluidic device will have a chamber with a wide, shallow central platform region and four surrounding wells. Two wells will be filled with buffer, one with protein solution, and one with a potential precipitant (e.g., sodium chloride). Diffusion from the wells into the platform region will create stable concentration gradients of protein and precipitant, and crystals will form only in regions where the two concentrations are above the saturation level. A three-dimensional microfluidic feed system will be constructed to allow multiple working units to operate in parallel. Once the crystallization is complete, the user will construct a light micrograph of the results, and the software component of the PDV be used. The software will solve the diffusion problem to determine the solution composition in the platform region, and then each pixel will be converted from (x,y) values to concentrations of protein and precipitant, and the corresponding points will be used to construct a phase diagram for the protein-precipitant system. Thus, a single experiment will generate the entire solution phase diagram. The phase diagram will then be available for use in a variety of phase-diagram-based methods for crystal growth.
The broad impact of the Phase Diagram Visualizer will be considerable. A conservative estimate of the human proteome is 100,000 proteins, and only a few thousand have been structurally characterized. Given that the major obstacle to protein structure characterization is growing a large, high quality crystal, access to phase diagram information will be a boon to structural biologists. Structural biology has impact not only on basic cell science but also on public health through understanding of disease and design of new drugs. The project will also contribute to Education and Human Resources through the training of a graduate student and an undergraduate who will work together, to their mutual benefit. A modified version of the PDV will make an excellent demonstration for use in undergraduate or secondary teaching, illustrating fundamental scientific concepts such as diffusion and solubility.
