Anachem Lipidic Cubic Phase Crystallization Robot

Anachem 脂质立方相结晶机器人

• 批准号: 7792043
• 负责人: Robert M Stroud
• 金额: $ 17.54万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-20 至 2012-02-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7792043
• 关键词: Adrenergic Receptor; Archaea; Color; Complex; Crystallization; Detergents; Economics; Environment; Evaluation; Genome; Grant; Harvest; Health; Human; Image; Joints; Laboratories; Lipid Bilayers; Lipids; Liquid substance; Membrane Lipids; Membrane Proteins; Methods; North America; Pharmaceutical Preparations; Phase; Porifera; Proteins; Resolution; Robot; Robotics; Science; Screening procedure; Simulate; Solutions; Structure; System; Therapeutic; X-Ray Crystallography; density; design; drug discovery; instrument; interfacial; nanolitre; success

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.

描述（由申请人提供）：脂质立方相中的膜蛋白结晶和结构测定或海绵状膜蛋白酶：膜蛋白占基因组的30%，并且是当今使用的所有药物的40%以上的靶点，然而仅在过去的一年中才出现了第一个真核细胞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，并极大地增强了针对这一未开发的治疗领域的药物发现。结构测定的手段是X射线晶体学，需要高质量的晶体膜蛋白。迄今为止，大多数结晶的膜蛋白已经使用去污剂将膜蛋白从其脂质环境中溶解出来而结晶。另一种方法是使用脂质的“中间相”使用更天然的脂质环境结晶。然而，直到最近，脂质立方相（LCP）方法仅涉及来自古细菌的小的有色蛋白质。一个更水合和开放的中间相，减少界面曲率，支持更大和更笨重的蛋白质容易结晶。这种方法已经产生了光捕获II复合物的结构级晶体，分辨率为2.45 º，具有比去污剂方法显著更高的堆积密度。为了便于使用这种方法进行快速，经济的筛选，Martin Caffrey博士将机器人系统用于这种方法的高通量应用，否则对于大多数膜蛋白来说是难以处理的。该机器人使用标准实验室流体处理机器人，其通过添加30纳升体积的粘性中间相的精确分配器进行修改，每次试验增加20 nL蛋白质和800 nL沉淀剂溶液。96个威尔斯可以设置使用机器人在9分钟。通过成像系统评价结晶进程，该成像系统能够在15分钟内对96孔板进行成像。通过初级赠款，我们每个人都生产纯均匀和稳定的膜蛋白校准结晶。到目前为止，Stroud小组已经确定了15种保持在洗涤剂中的膜蛋白的结构。这个机器人将允许膜脂质中膜蛋白的不同类型的结晶。该方法已被Scripps的Stevens小组使用，取得了里程碑式的成功，使用Caffrey的机器人，导致2-肾上腺素能受体的结构（Cherezov，V.，等人，Science 318：1258-65，2007）。Caffrey为Anachem提供设计，并与我们合作共同更新路线图提案GM 73210。到目前为止，只有两个这样的仪器在使用;这将是第三个，在马丁·卡夫瑞的指导下，由Anachem有限公司建造，由Zinsser北美公司分销。这个机器人使我们能够以高通量的形式利用最有前途的膜蛋白结晶新方法。因此，它打开了在模拟自然环境（脂质双层）的精确条件下结晶更多膜蛋白的视野。 健康相关性：Lipidic Cubic Phase机器人仪器有助于膜蛋白在其脂质环境中的结构测定。膜蛋白占所有真核生物蛋白质的约30%，并且是当今使用的所有药物的40%以上的靶点，然而仅在过去的一年中才出现了第一个真核生物膜蛋白与药物的结构相互作用。该提案寻求推进膜蛋白的一般结构测定，并且靶向对人类健康重要的人类或病原性膜蛋白。