HUMAN PLASMA PLATELET ACTIVATING FACTOR ACETYLHYDROLASE

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。血浆形式的人血小板活化因子乙酰水解酶（pPAFAH）通过降低PAF水平作为一般抗炎清除剂起作用，并且与过敏性休克、哮喘和过敏反应有关。pPAFAH是一种膜相关蛋白，没有已知的同源物，是一个有价值的结构靶标。 天然酶已在各种条件下用各种去污剂结晶。我们目前有高质量的晶体（60-100微米）的活性蛋白（图1，附件）。到目前为止，最好的晶体在我们的内部仪器上衍射到3.2 A的分辨率（图2），镶嵌度优于0.7度。活性蛋白质也在硫酸铵中结晶并衍射至约3.5 A的分辨率，但该晶体（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位点定向灭活剂对氧磷的复合物，其中其活性位点丝氨酸被共价修饰。这些失活蛋白质的晶体（图3）衍射到约3.5-4.0 A（图4），但同样高度镶嵌。最终，我们想追求这两种晶体形式，因为它是非常重要的，知道两个配体自由和活性位点结合形式的酶的结构。 由于pPAFAH没有同源结构，我们希望收集MAD数据，以解决我们的结构相。报告表明蛋白质的Br浸泡适合于MAD定相。最近，在8月份我们实验室束流时间的剩余时间内，在APS束线14-ID-B上筛选了pPAFAH的溴化物浸泡晶体。如图5所示，该晶体显示出清晰的衍射，分辨率为3.4 A，并且在30分钟的X射线暴露期间没有遭受明显的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相信，我们将能够获得高质量的X射线衍射，用于MAD定相和精确地解决高分辨率的天然结构。

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.