CRYSTALLOGRAPHIC ANALYSES OF UNLIGANDED GIANT ANNELID RESPIRATORY 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来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 细胞外环节动物呼吸蛋白组装成大的复合物（高达3,600 kDa），这使得氧结合能力的实质性浓度成为一个单一的颗粒。 这些蛋白质由于其大尺寸而表现出高度合作的氧结合特性，这取决于二价阳离子和质子的相互作用。 该建议的目标是研究这些大的呼吸蛋白的强变构特性的结构基础。 我们的研究对象是三种不同的，但相关的，合作的环节动物复合体。 其中包括两个3 600 kDa的组合，即来自蚯蚓和Arenicola marina的erythrocruorins和在热液喷口管虫Riftia pachyptila中发现的400 kDa血红蛋白。 我们已经确定了与结合配体（无论是氧或一氧化碳）的每个复合物的晶体结构，使用从BioCARS光束线14-BMC收集的衍射数据。 这两个3,600 kDa的红细胞cruorins的晶体结构揭示了180多肽链，形成六边形双层结构，但改变了安排的六边形层源于异源三聚体卷曲螺旋结构域的中心附近的每个复杂的。 400 kDa Riftia复合物结构是24个亚基的中空D3对称球体，其中每一半类似于在红血球中发现的十二聚体复合物排列（弗洛雷斯等人，PNAS 102，2713-2718，2005）。 目前还没有可用于该呼吸蛋白家族的任何成员的无配体晶体结构。 我们最近在没有配体的情况下生长了这些蛋白质的晶体。 这些蛋白质的晶体结构将使我们能够理解介导协同性的配体连接的结构转变，它们的比较将揭示每个相似但不同的四元排列影响配体连接转变范围的程度，以及这与功能特性的相关性。

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.