IDBR: Closed-Loop Protein Crystallization

布兰迪斯大学的弗雷登博士获得了一项资助，用于开发用于蛋白质结晶的微流控工具。尽管蛋白质结晶的基本机制得到了研究界的一致同意，但标准的结晶实践并不包括以最佳方式利用结晶背后的物理原理的方法和工具。为了克服目前结晶技术的不足，Fraden博士将开发闭环式蛋白质结晶器(CLC)，它结合了微流体的高通量、精确度和低体积等特性。CLC的概念基础是增加过饱和度，直到检测到纳米级晶核，然后动态改变过饱和条件来控制晶体生长速度。微流控蛋白质结晶装置(CLC)由一系列孔组成，用于存储数百纳升体积的含有蛋白质溶液的液滴。该设备包括一个半透膜，允许蛋白质溶液的浓度变化，以及一个温度阶段，允许在不同的井中的液滴经历不同的温度。CLC将包括动态光散射(DLS)，它将用于检测成核开始和随后的晶体生长。DLS的输出将用于闭环反馈模式，以控制结晶形成时刻的浓度和温度的热力学变量，这是控制结晶的最佳时刻。结构生物学领域致力于解决蛋白质的结构问题，因为人们相信结构意味着功能。结构？功能关系对于包括新生物分子在内的新材料的基础知识仿生工程具有重要意义。蛋白质的结构通常是通过X射线衍射法获得的，而结晶是这一过程中的一个必要步骤。组成人类基因组的30,000种蛋白质中有三分之一存在于膜上，但只有一种人类膜蛋白的结构得到了解决。结晶是这一过程中的瓶颈，本项目的目标是通过开发闭环式结晶器来消除这一瓶颈。此外，该项目涉及的尖端芯片实验室仪器将为对美国经济和科学基础设施至关重要的技术部门的学生提供培训。

A grant has been awarded to Dr. Fraden at Brandeis University to develop microfluidic tools to crystallize proteins. Although the basic mechanism of protein crystallization is agreed upon by the research community, standard crystallization practices do not incorporate methods and instruments that optimally exploit the physical principles underlying crystallization. To overcome the deficiencies of current crystallization technologies Dr. Fraden will develop the Closed-Loop Protein Crystallizer (CLC), which incorporates the attributes of high-throughput, precision, and low volume that are characteristics of microfluidics. The conceptual underpinning of the CLC is to increase supersaturation until nanometer scale crystal nuclei are detected and then to dynamically vary the supersaturation conditions to control the rate of crystal growth.The microfluidic protein crystallization device (CLC) consists of an array of wells to store hundreds of nanoliter volume drops containing protein solution. The device incorporates a semi-permeable membrane that allows the concentration of the protein solution to be varied, as well as a temperature stage that allows drops in separate wells to experience different temperatures. The CLC will incorporate Dynamic Light Scattering (DLS) which will be used to detect the onset of nucleation and subsequent crystal growth. The output of the DLS will be used in a closed-loop feedback mode to control the thermodynamic variables of concentration and temperature at the moment of crystallogenesis, which is the optimal moment to control crystallization. The field of Structural Biology is dedicated to solving the structure of proteins motivated by the belief that ?structure implies function?. Structure ? function relationships are important for fundamental knowledge biomimetic engineering of new materials including new biomolecules. Protein structure is most commonly obtained through x-ray diffraction and crystallization is a necessary step in this process. One third of the 30,000 proteins that comprise the human genome reside in the membrane, yet only one human membrane protein has had its structure solved. Crystallization is the bottleneck in this process and the goal of this project is to eliminate this bottleneck by the development of the Closed Loop Crystallizer. Additionally, the cutting edge lab-on-a-chip instrumentation involved in this project will provide training to students in a technology sector vital to the US economy and scientific infrastructure.