CRYSTALLOGRAPHIC ANALYSES OF UNLIGANDED GIANT ANNELID RESPIRATORY PROTEINS

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。 细胞外环节动物呼吸蛋白组装成大的复合体(高达3600 kDa)，这使得大量的氧结合能力形成一个单一的颗粒。与它们的大尺寸相匹配，这些蛋白质表现出高度合作的氧结合特性，这依赖于二价阳离子和质子的相互作用。这项提议的目的是研究这些大型呼吸蛋白具有强大变构特性的结构基础。 我们的主题是三个截然不同但又相互关联的合作环节动物复合体。其中包括两个3600 kDa的组合，来自Lumbricus terrestris和Arenicola marina的红血球蛋白，以及在热液喷口管虫Riftia pachyptila中发现的400 kDa的血红蛋白。我们利用BioCARS光束线14-BMC的衍射数据确定了每个带有结合配体(氧或一氧化碳)的配合物的晶体结构。这两个3,600 kDa的红血球蛋白的晶体结构都显示了180个多肽链，形成了六方双层结构，但六方层的排列发生了变化，这是由于每个复合体中心附近的异三聚螺旋卷曲结构域的差异造成的。400 kDa的Riftia复合体结构是一个由24个亚基组成的中空D3对称球体，每一半的排列类似于在红血球蛋白中发现的十二聚体复合体(Flores等人，PNAS 102,2713-2718,2005)。目前，这个呼吸蛋白家族中的任何成员都没有可用的未连接晶体结构。我们最近在没有配体的情况下生长了每一种蛋白质的晶体。这些蛋白质的晶体结构将使我们能够理解介导协作性的配体连接的结构跃迁，它们的比较将揭示每个相似但不同的四元排列对配体连接跃迁范围的影响程度，以及这与功能性质的相关性。

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. Extracellular annelid respiratory proteins assemble into large complexes (up to 3,600 kDa), which allows substantial concentration of oxygen binding capacity into a single particle. Befitting their large size, these proteins exhibit highly cooperative oxygen binding properties that are dependent upon the interplay of divalent cations and protons. The goal of this proposal is to investigate the structural basis for the strong allosteric properties of these large respiratory proteins. Our subjects are three distinct, but related, cooperative annelid complexes. These include two 3,600 kDa assemblages, the erythrocruorins from Lumbricus terrestris and Arenicola marina and a 400 kDa hemoglobin found in the hydrothermal vent tubeworm, Riftia pachyptila. We have determined crystal structures of each complex with bound ligand (either oxygen or carbon monoxide), using diffraction data collected from BioCARS beamline 14-BMC. The crystal structures of both 3,600 kDa erythrocruorins reveal 180 polypeptide chains that form hexagonal bilayer structures, but with altered arrangements of the hexagonal layers stemming from differences in heterotrimeric coiled-coil domains near the center of each complex. The 400 kDa Riftia complex structure is a hollow D3 symmetric sphere of 24 subunits, with each half arranged similarly to dodecamer complexes found in the erythrocruorins (Flores et al., PNAS 102, 2713-2718, 2005). There are currently no unliganded crystal structures available for any members of this family of respiratory proteins. We have recently grown crystals of each of these proteins in the absence of ligand. Crystal structures of these proteins will allow us to understand the ligand-linked structural transitions that mediate cooperativity and their comparison will reveal the degree to which each similar, but distinct, quaternary arrangement impacts the range of ligand-linked transitions and how this correlates with the functional properties.