Anachem Lipidic Cubic Phase Crystallization Robot

描述(申请人提供)：膜蛋白在脂质立方相或海绵中间相中的结晶和结构测定：膜蛋白占基因组的30%，是目前使用的所有药物的靶标，然而直到过去一年才出现第一个真核膜蛋白与药物的结构相互作用。原子级结构定义了作用机制，并极大地促进了针对这一未开发的治疗前景的药物发现。结构测定的手段是X射线结晶学，需要高质量的结晶膜蛋白。到目前为止结晶的大多数膜蛋白都是使用洗涤剂将膜蛋白从其脂质环境中溶解出来的。另一种方法是在更自然的脂类环境中使用脂类的“中间相”进行结晶。然而，直到最近，脂质立方相(LCP)方法仅适用于来自古细菌的小分子有色蛋白质。界面曲率减小的更多水合和开放的中间相支持更大和更大的蛋白质的容易结晶。这种方法已经产生了结构级的捕光II络合物晶体，分辨率达到2.45？，其堆积密度明显高于洗涤剂方法。为了便于使用这种方法进行快速、经济的筛查，Martin Caffrey博士调整了机器人系统，以适应这种方法的高通量应用，否则这种方法对大多数膜蛋白来说是难以解决的。该机器人使用标准的实验室流体处理机器人，在每次试验中增加了一个精确的30纳升体积的粘性中间相分配器，并增加了20毫升蛋白质和800毫升沉淀剂溶液。使用该机器人可以在9分钟内打好96口井。结晶进度的评估是通过成像系统实现的，能够在15分钟内对96孔板进行成像。通过初级资助，我们每一家都生产纯净、均匀和稳定的膜蛋白，这些蛋白经过结晶校准。到目前为止，Stroud小组已经确定了洗涤剂中保持的15种膜蛋白的结构。这个机器人将允许膜脂中的膜蛋白进行不同类型的结晶。这种方法已经被斯克里普斯的史蒂文斯小组使用卡弗里的机器人获得了里程碑式的成功，这导致了2-肾上腺素能受体的结构(Cherezov，V.，et al.科学318：1258-65,2007)。卡弗里为Anacem提供设计，也是我们联合更新路线图提案GM 73210的合作者。到目前为止，只有两台这样的仪器在使用；这将是第三台，在马丁·卡弗里的指导下，由Anacem，Ltd制造，由Zinsser North America，Inc.分销。这个机器人使我们能够以高通量格式利用最有前途的膜蛋白结晶的新方法。因此，它打开了在模拟自然环境的精确条件下结晶更多膜蛋白的视野，即脂双层。 与健康相关：脂类立方相机器人仪器有助于膜蛋白在其脂类环境中的结构测定。膜蛋白约占真核生物蛋白质的30%，是目前使用的药物的靶标，但直到最近一年才出现了第一个真核膜蛋白与药物的结构相互作用。这项建议旨在推进膜蛋白的总体结构测定，并针对对人类健康重要的人类或致病性膜蛋白。

DESCRIPTION (provided by applicant): Membrane Protein Crystallization and Structure Determination in Lipidic Cubic Phase or Sponge Mesophase: Membrane proteins constitute 30% of the genome, and are targets for over 40% of all drugs in use today, however only in the past year have structural interactions of the first eukaryotic membrane protein with a drug emerged. Atomic level structures define mechanisms of action, and greatly enhance drug discovery against this untapped therapeutic landscape. The means of structure determination is X-ray crystallography requiring high quality crystalline membrane proteins. Most of the membrane proteins crystallized to date have been crystallized using detergents to solubilize the membrane protein out of its lipid environment. An alternative approach is to crystallize using more natural lipid environments using 'mesophases' of lipids. However until recently the Lipidic Cubic Phase (LCP) methods pertained only to small colored proteins from archaebacteria. A more hydrated and open mesophase, of reduced interfacial curvature supports facile crystallization of larger and bulkier proteins. This method has yielded structure- grade crystals of the light-harvesting II complex to 2.45 ¿ resolution with dramatically higher packing density than by detergent methods. To facilitate rapid, economic screening using this method, Dr. Martin Caffrey adapted a robotic system to high throughput application of this method that is otherwise intractable for the majority of membrane proteins. The robot uses a standard laboratory fluid handling robot modified by addition of an accurate dispenser of viscous mesophases in 30 nanoliter volumes augmented by 20 nL protein and 800 nL precipitant solution for each trial. 96 wells can be set up using the robot in 9 minutes. Evaluation of crystallization progress is by an imaging system enabling a 96-well plate to be imaged in 15 minutes. Through primary grants we each produce pure homogeneous and stable membrane proteins calibrated for crystallization. To date the Stroud group has determined structures of 15 membrane proteins maintained in detergent. This robot will allow a different type of crystallization of membrane proteins in membrane lipids. The method has been used with landmark success by the Stevens group at Scripps, using Caffrey's robot, that led to structures of 2-adrenergic receptors (Cherezov, V., et al. Science 318: 1258-65, 2007). Caffrey provided designs to Anachem and is a collaborator with us on our joint renewal of Roadmap proposal GM 73210. To date there are only two of these instruments in use; this would be a third, built under the guidance of Martin Caffrey, by Anachem, Ltd and distributed by Zinsser North America, Inc. This robot enables us to tap into the most promising new method of crystallization of membrane proteins in a high throughput format. As such it opens the horizon to crystallize many more membrane proteins under defined exact conditions that simulate the natural environment, the lipid bilayer. HEALTH RELEVANCE: The Lipidic Cubic Phase robotic instrument facilitates structure determination of Membrane Proteins in their lipid environments. Membrane proteins constitute ~30% of all eukaryotic proteins and are targets for over 40% of all drugs in use today, however only in the past year have structural interactions of the first eukaryotic membrane protein with a drug emerged. This proposal seeks to advance structural determination of membrane proteins in general, and targets human or pathogenic membrane proteins of importance to human health.