IDBR: Phase Diagram Visualizer (PDV) for Biomacromolecules

该奖项是对开发一种名为相图显像仪(PDV)的新仪器的奖励。PDV将用于优化蛋白质结晶的条件。这具有很大的潜在价值，因为目前蛋白质结晶的能力是确定蛋白质结构的瓶颈。PDV是一种新型微流控装置，用于产生蛋白质、沉淀剂和缓冲液的重叠浓度梯度。晶体的形成将被可视化，软件将根据蛋白质和其他溶质的扩散常数确定体系的相图。PDV的微流控装置将有一个腔室，其中有一个宽而浅的中央平台区域和四个周围的井。两个井将填充缓冲液，一个井填充蛋白质溶液，一个井填充潜在沉淀剂(例如氯化钠)。从井向平台区域的扩散将产生稳定的蛋白质和沉淀剂的浓度梯度，只有在这两种浓度高于饱和水平的区域才会形成晶体。将建造一个三维微流控进给系统，以允许多个工作单元并行运行。一旦结晶完成，用户将构建结果的光学显微图像，并使用PDV的软件组件。该软件将求解扩散问题来确定平台区域内的溶液组成，然后将每个像素从(x，y)值转换为蛋白质和沉淀剂的浓度，并使用对应的点来构建蛋白质-沉淀剂体系的相图。因此，一次实验就可以生成整个溶液相图。相图将可用于各种基于相图的晶体生长方法。相图展示器的广泛影响将是相当大的。据保守估计，人类蛋白质组有10万种蛋白质，只有几千种蛋白质的结构特征得到了鉴定。鉴于蛋白质结构表征的主要障碍是生长出大而高质量的晶体，获得相图信息将是结构生物学家的福音。结构生物学不仅对基础细胞科学有影响，而且通过对疾病的了解和新药的设计对公众健康也有影响。该项目还将通过培训一名研究生和一名本科生来促进教育和人力资源，他们将共同努力，使他们互惠互利。PDV的修改版本将成为本科或中学教学中使用的一个很好的演示，说明基本的科学概念，如扩散和溶解度。

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.