STRUCTURAL BIOLOGY OF MEMBRANE PROTEINS

膜蛋白的结构生物学

• 批准号: 8363518
• 负责人: MICHAEL G MALKOWSKI
• 金额: $ 2.45万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363518
• 关键词: Animals; Carbon; Caribbean region; Cells; Cytochromes; Data Collection; Detergents; Environment; Enzymes; Funding; Grant; Human; Invertebrates; Laboratories; Light; Lipoxygenase; Membrane; Membrane Proteins; Mus; National Center for Research Resources; Oryza sativa; Oxygen; Oxygenases; Plants; Polyunsaturated Fatty Acids; Principal Investigator; Production; Prostaglandin-Endoperoxide Synthase; Proteins; Research; Research Infrastructure; Resolution; Resources; Roentgen Rays; Source; Synchrotrons; United States National Institutes of Health; beamline; coral; cost; 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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 Malkowski实验室的研究重点是了解不同类别的膜酶如何利用立体选择性氧化作用从一组定义的多不饱和脂肪酸（PUFA）底物中产生独特的氧脂。 氧脂素是在植物和动物中发现的生物活性脂质介质，其分别经由细胞色素P450、脂氧合酶（LOX）和环加氧酶（考克斯）的催化活性通过添加一个、两个或四个氧分子由18-22碳PUFA生物合成。 衍射研究集中在植物、动物和无脊椎动物中参与氧脂素生产的三种不同的膜整合酶：a）来自水稻的病原体诱导加氧酶（PIOX）; B）人类和小鼠环氧合酶-2（考克斯-2）;和c）来自加勒比海珊瑚Plexaura homomalla的环氧合酶。 在国家同步加速器光源（光束线X4 A; 2005年3月）进行的考克斯-2的初始衍射实验证实晶体实际上是蛋白质;然而，仅观察到6.5分辨率的衍射。 这个项目需要同步辐射有两个主要原因。 首先，当考克斯-2晶体长大时，它们对实验室X射线源的作用很弱，只有8-10埃。 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都很敏感，使得处理这些晶体以收集数据变得困难。 通常许多晶体必须筛选，以找到一个保持最小的镶嵌性（<2 <$a）和合理的衍射。