HUMAN PLASMA PLATELET ACTIVATING FACTOR ACETYLHYDROLASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 血浆形式的人血小板活化因子乙酰水解酶(PPAFAH)通过降低PAF水平起到一般抗炎清除剂的作用，并与过敏性休克、哮喘和过敏反应有关。作为一种膜相关蛋白，pPAFAH没有已知的同源物，是一个有价值的结构靶点。 天然酶在不同的条件下与各种洗涤剂结晶。我们目前拥有高质量的活性蛋白质晶体(60-100微米)(附图1)。到目前为止，最好的晶体已经在我们的内部仪器(图2)上衍射到3.2A的分辨率，马赛克优于0.7度。活性蛋白质也在硫酸铵中结晶，衍射率约为3.5A，但这种晶体(100-120微米)是高度镶嵌的。在另一种方法中，我们已经结晶了酶与活性部位定向的灭活剂对氧磷的复合体，其中其活性部位丝氨酸是共价修饰的。这些失活蛋白的晶体(图3)衍射率约为3.5-4.0A(图4)，但同样是高度镶嵌的。最终，我们想要追求这两种晶型，因为了解酶的配体自由形式和活性部位结合形式的结构是非常重要的。 由于pPAFAH没有同源结构，我们希望收集MAD数据以解决我们的结构的相。报告表明，蛋白质的BR浸泡适合于疯狂相变。最近，在8月份我们实验室剩余的光束时间里，在APS光束线14-ID-B上筛选到了一个溴化物浸泡的pPAFAH晶体。如图5所示，该晶体表现出清晰的衍射，分辨率为3.4A，在30分钟的X射线曝光中没有受到明显的X射线损伤。 我们的初步结果加上大量新鲜晶体的供应，使我们有信心能够获得高质量的X射线衍射，用于MAD相变和随后将自然结构解算到高分辨率。

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 plasma form of human platelet activating factor acetylhydrolase (pPAFAH) functions by reducing PAF levels as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma and allergic reactions. As a membrane associated protein with no known homologues, pPAFAH is a worthy structural target. Native enzyme has been crystallized in various conditions with a variety of detergents. We currently have high quality crystals (60-100 microns) of active protein (Fig. 1, attached). The best crystals so far have diffracted to a resolution of 3.2 A on our in-house instrument (Fig. 2) with a mosaicity better than 0.7 degrees. The active protein also crystallized in ammonium sulfate and diffracted to a resolution of about 3.5 A, but this crystal (100-120 microns) was highly mosaic. In an alternate approach, we have crystallized a complex of the enzyme with the active site directed inactivator paraoxon, where its active site serine is covalently modified. These crystals (Fig. 3) of inactivated protein diffracted to about 3.5-4.0 A (Fig. 4) but were likewise highly mosaic. Ultimately we would like to pursue both crystal forms, as it is very important to know the structures of both ligand free and active site bound forms of the enzyme. Since pPAFAH does not have a homologous structure, we would like to collect MAD data in order to solve the phases for our structure. Reports suggest Br soaks of proteins are suitable for MAD phasing. Recently a bromide soaked crystal of pPAFAH was screened during the remainder of our lab beam time in August at the APS beamline 14-ID-B. As shown in Fig. 5, this crystal showed clean diffraction to a resolution of 3.4 A and did not suffer appreciable X-ray damage during the 30 min of X-ray exposure. Our preliminary results together with an abundant supply of fresh crystals makes us confident that we will be able to obtain high quality X-ray diffraction for MAD phasing and subsequantly solving the native structure to high resolution.