RECOGNITION BY RECEPTORS

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。 病原体和宿主之间相互作用的特殊性允许入侵(当病原体识别宿主时)和免疫识别(当宿主识别病原体时)。这些项目研究这两种类型的相互作用，既包括膜跨越区域，也包括分离的蛋白质可溶性区域。最初的结构尚未公布。 第一个项目包括人类Toll样受体(TLRs)和细菌外膜孔蛋白之间的相互作用。我们已经生长了作为TLR配体的细菌孔蛋白的初始晶体，其衍射率可达2.3？这些晶体在三个维度中的两个维度上都很小，以至于最多具有可调波长(&gt；0.1 mm)的同步加速器的大光束尺寸限制了我们的数据质量。我们用自己的发电机看不到任何衍射。由于这些孔蛋白与已知结构的孔蛋白没有显著的序列相似性，分子置换失败。这种蛋白质没有足够的蛋氨酸来执行Se-Met MAD，我们正在收集衍生品。从迄今收集的50个数据集中，我们确定了两个衍生品。第一种是单位的Lu导数，在此过程中，我们建议对浸渍Lu的晶体进行全4波长的MAD，以改善位相。二阶衍生物是具有4个位置的碘化物衍生物。这些晶体的衍射率很低(4A分辨率)，我们建议采用不同浸泡浓度和次数的晶体，以将衍射极限提高到3.5A以下的分辨率。此外，我们还将带来其他几个重原子浸泡的晶体(见上)。 第二个项目研究相反的病原体识别现象--即细菌受体对宿主唾液酸的识别。细菌受体(GspB)的识别区域已经生长出晶体。像孔蛋白晶体一样，这些晶体生长成细棒，缺乏足够的蛋氨酸来通过Se-Met确定结构。此外，我们认为这些晶体的小尺寸排除了在我们的家用发电机上观察到任何衍射的可能性。我们正在开始寻找重原子的衍生物；尽管已经从浸泡了5种不同化合物的晶体中收集了数据，但还没有鉴定出任何衍生物。我们将带来天然蛋白质，以确定较小的波束尺寸是否会提高数据集的质量，此外，我们还将带来上述衍生品浸泡，用于MAD数据收集。 最后的项目是完整膜细菌复合体II的同系物喹酚：富马酸还原酶。这些晶体长成长棒。在家用发电机上收集的最佳数据集是6A分辨率，而在同步加速器上收集的最佳数据集是2.7A分辨率。我们目前的项目是确定与伙伴蛋白的共结构。到目前为止，文献中只报道了膜蛋白与伴侣蛋白形成的复合体中的一种共结构。因此，从技术发展的角度来看，这一结构的意义将是巨大的。此外，这个复合体的生物学意义是巨大的。

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 specificity of interactions between pathogen and host allows both invasion (when the pathogen recognizes the host) and immunological recognition (when the host recognizes the pathogen. These projects investigate both types of interactions, and include both the membrane-spanning region as well as the separated soluble region of the proteins. The initial structures have not yet been published. The first project encompasses the interaction between human toll like receptors (TLRs) and bacterial outer membrane porins. We have grown initial crystals of the bacterial porins that are TLR ligands that diffract to 2.3 ¿ resolution. These crystals are small in two of the three dimensions such that the large beam size at most synchrotrons with tunable wavelengths (>0.1 mm) limits the quality of our data. We are unable to see any diffraction using our home generator. Since these porins do not have significant sequence similarity to porins of known structure, molecular replacement has failed. The protein does not have sufficient methionines to perform Se-Met MAD and we are collecting derivatives. From 50 data sets collected so far, we have identified two derivatives. The first is a Lu derivative with a single site, and during this beamtime, we propose to do a full 4-wavelength MAD on Lu-soaked crystals in order to improve the phases. The second derivative is an iodide derivative with 4 sites. These crystals diffracted poorly (4 A resolution) and we propose bringing crystals that vary the soaking concentration and times in order to improve the diffraction limit to better than 3.5 A resolution. In addition, we will bring several other heavy atom soaked crystals (see above). The second project investigates the opposite pathogenic recognition phenomenon - i.e. the recognition of host siaylic acids by the bacterial receptor. Crystals have been grown of the recognition region of the bacterial receptor (GspB). Like the porin crystals, these grow as thin rods and lack sufficient methionines to determine the structure by Se-Met. In addition, we believe that the small size of these crystals is precluding observation of any diffraction on our home generator. We are at the beginning of searching for heavy atom derivatives; although data have been collected from crystals soaked with 5 distinct compounds, no derivatives have yet been identified. We will bring native protein to determine if a smaller beam size will improve the quality of the data set, and in addition, we will bring the above mentioned derivative soaks for MAD data collection. The final project is of the integral-membrane bacterial complex II homolog quinol:fumarate reductase. These crystals grow as long rods. The best data set collected on the home generator is at 6A resolution, while the best data set collected at the synchrotron is at 2.7 A resolution. Our current project is to determine a co-structure with a partner protein. Only one co-structure of membrane proteins in complex with a partner protein has been reported in the literature to date. Thus the significance of this structure from a technique development standpoint would be great. In addition, the biological significance of this complex is immense.