STRUCTURAL BIOLOGY OF MEMBRANE PROTEINS

膜蛋白的结构生物学

• 批准号: 8171494
• 负责人: MICHAEL G MALKOWSKI
• 金额: $ 2.82万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171494
• 关键词: Animals; Carbon; Caribbean region; Cells; Computer Retrieval of Information on Scientific Projects Database; Cytochromes; Data Collection; Detergents; Environment; Enzymes; Funding; Grant; Human; Institution; Invertebrates; Laboratories; Light; Lipoxygenase; Membrane; Membrane Proteins; Mus; Oryza sativa; Oxygen; Oxygenases; Plants; Polyunsaturated Fatty Acids; Production; Prostaglandin-Endoperoxide Synthase; Proteins; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Source; Synchrotrons; United States National Institutes of Health; beamline; coral; cyclooxygenase 2; lipid mediator; pathogen; research study; structural biology; synchrotron radiation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The research focus in the Malkowski laboratory is to understand how different classes of integral membrane enzymes utilize stereoselective oxygenations to generate unique oxylipins from a defined set of polyunsaturated fatty acid (PUFA) substrates. Oxylipins are bioactive lipid mediators found in plants and animals that are biosynthesized from 18-22 carbon PUFAs through the addition of one, two, or four molecules of oxygen via the catalytic activity of cytochrome P450s, lipoxygenases (LOX), and cyclooxygenases (COX), respectively. Diffraction studies are focused on three different integral membrane enzymes involved in oxylipin production in plants, animals, and invertebrates: a) pathogen-inducible oxygenase (PIOX) from Oryza sativa; b) human and murine cyclooxygenase-2 (COX-2); and c) a cyclooxygenase enzyme from the Caribbean coral Plexaura homomalla. Initial diffraction experiments of COX-2, carried out at the National Synchrotron Light Source (beamline X4A; March 2005), confirm that the crystals are in fact protein; however, diffraction was only observed to 6.5¿ resolution. This project requires synchrotron radiation for two major reasons. First, while crystals of COX-2 grow large, they diffract only weakly to 8-10 ¿ on laboratory X-ray sources. Second, their relatively empty unit cell, combined with their sensitivity to any change in the detergent environment, make handling these crystals for data collection difficult. Often many crystals must be screened to find one that maintains minimal mosaicity (<2¿a) and reasonable diffraction.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 Malkowski 实验室的研究重点是了解不同类别的整合膜酶如何利用立体选择性氧化从一组确定的多不饱和脂肪酸 (PUFA) 底物中生成独特的氧脂素。 Oxylipins 是在植物和动物中发现的生物活性脂质介质，由 18-22 碳 PUFA 生物合成，分别通过细胞色素 P450、脂氧合酶 (LOX) 和环加氧酶 (COX) 的催化活性添加一分子、两分子或四分子氧。 衍射研究主要集中在植物、动物和无脊椎动物中参与氧脂素生产的三种不同的整合膜酶：a) 来自稻的病原体诱导加氧酶 (PIOX)； b) 人和鼠环氧合酶-2 (COX-2)； c) 来自加勒比珊瑚 Plexaurahomalla 的环氧合酶。 在国家同步加速器光源（光束线 X4A；2005 年 3 月）进行的 COX-2 初始衍射实验证实晶体实际上是蛋白质；然而，仅在 6.5° 分辨率下观察到衍射。 该项目需要同步加速器辐射有两个主要原因。 首先，虽然 COX-2 晶体长大，但它们在实验室 X 射线源上的衍射仅微弱至 8-10 ¡ 其次，它们相对空的晶胞，加上它们对洗涤剂环境的任何变化的敏感性，使得处理这些晶体进行数据收集变得困难。 通常必须对许多晶体进行筛选，以找到一种保持最小镶嵌度（<2¿a）和合理衍射的晶体。