Investigating structural diversity of salivary lipocalins of bloodsucking insects

描述（由申请人提供）：我的项目的总体目标是开发和测试方法，以靶向蛋白质进行表征，其序列介于一对具有不同结构和功能特性的相关蛋白质之间。特别地，兴趣福尔斯落在确定唾液脂质运载蛋白的医学相关类别内的结构变化上。这些蛋白质存在于几种吸血昆虫的唾液中，它们是已知的Chagas1疾病的载体。Triabin是一种具有相似生物学功能但不同拓扑结构的相关蛋白质，其鉴定促使人们对其独特结构的机制进行研究。为此，我们已经确定了三个序列的中间脂质运载蛋白的连接triabin的nitrophorin 2，一个相关的脂质运载蛋白与已知的结构和功能。该研究的目的是开发用于克隆、表达和纯化这些序列中间体脂质运载蛋白的方案。我研究的第二个目标是通过核磁共振和X射线晶体学技术确定其中至少一种的结构。一个组氨酸标记的序列中间体已被克隆，表达，重折叠和纯化，并已证明自己适合于研究核磁共振。另外两种蛋白质处于表达、重折叠和纯化的不同阶段。它们也将被带沿着进行结构研究，但优先级较低。此外，我将开发一个协议来纯化和重折叠未标记的变体。标记的和未标记的变体之间的2D相关（HSQC）光谱的比较对于评估标记的形式是否显示与天然结构一致的结构性质将是至关重要的。这项研究的可能结果将是序列中间唾液脂质运载蛋白的结构测定。有了这个结构，我们将能够更准确地确定有助于triabin和nitrophorin 2之间结构差异的序列要求，并了解这个家族中的一些结构变异。了解蛋白质的结构和功能变异对于设计治疗靶点、确定寄生虫如何与宿主相互作用以及识别有可能形成疾病状态的蛋白质非常重要。我们正在开发和评估一种方法的目标识别蛋白质的序列之间的已知不同的属性。这项研究的首要目标是提高我们用结构和功能信息注释基因产物的能力。

DESCRIPTION (provided by applicant): The overall goal of my project is to develop and test methods to target proteins for characterization whose sequences are intermediate between a pair of related proteins with divergent structural and functional properties. In particular, interest falls in determining structural variation within the medically relevant class of salivary lipocalins. These proteins are found in the saliva of several bloodsucking insects, which are known vectors of Chagas1 disease. Identification of triabin, a related protein with similar biological function but a different topology, has prompted investigation into the mechanism which underlies its unique structure. To this end we have identified three sequence intermediate lipocalins which link triabin to nitrophorin 2, a related lipocalin with known structure and function. The goal of the proposed research is to develop protocols for cloning, expressing, and purifying these sequence intermediate lipocalins. The second aim of my research will be to determine the structure of at least one of them by NMR and x-ray crystallographic techniques. One histidine-tagged sequence intermediate has been cloned, expressed, refolded, and purified, and has proven itself amenable to studying by NMR. The other two proteins are in different stages of expression, refolding, and purification. They will also be brought along for structural studies, but at a lower priority. Additionally, I will develop a protocol to purify and refold untagged variants. Comparison of 2D correlation (HSQC) spectra between the tagged and the untagged variants will be critical for assessing whether the tagged forms display structural propertied consistent with a native structure. The likely outcome from this research will be structural determination of a sequence intermediate salivary lipocalin. With this structure, we will be able to more accurately determine the sequence requirements that contribute to the structural differences seen between triabin and nitrophorin 2, and understand some of the structural variation seen within this family. Understanding protein structural and functional variation is important in designing therapeutic targets, determining how parasites interact with hosts, and identifying proteins which have the potential to form into disease states. We are developing and assessing a method of target identification of proteins whose sequences are intermediate between those of known divergent properties. The overarching goal of this research is to increase our ability to annotate gene products with structural and functional information.