STRUCTURAL BIOLOGY OF MEMBRANE PROTEINS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，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。

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.