Investigating structural diversity of salivary lipocalins of bloodsucking insects

描述(申请人提供)：我的项目的总体目标是开发和测试目标蛋白质的方法，以确定其序列是介于两个结构和功能特性不同的相关蛋白质之间的中间序列。特别是，人们感兴趣的是确定与医学相关的唾液脂钙蛋白类别内的结构变异。在几种吸血昆虫的唾液中发现了这些蛋白质，这些昆虫是长崎病的已知媒介。Triabin是一种生物学功能相似但拓扑结构不同的相关蛋白质，其鉴定促使人们对其独特结构的机制进行了研究。为此，我们已经确定了三个连接Triabin和Nitrophorin 2的中间Lipocalin序列，Nitrophorin 2是一个已知结构和功能的相关Lipocalin。这项拟议研究的目标是开发克隆、表达和纯化这些序列中间Lipocalins的方案。我研究的第二个目标将是通过核磁共振和X射线结晶学技术确定至少其中一个的结构。一个组氨酸标记的序列中间体已经被克隆、表达、复性和纯化，并被证明适合于核磁共振研究。另外两种蛋白质处于不同的表达、复性和纯化阶段。他们也将被带去进行结构研究，但优先程度较低。此外，我将开发一个协议来净化和重新折叠未标记的变体。标记和未标记变体之间的二维相关(HSQC)谱的比较对于评估标记形式是否表现出与天然结构一致的结构特性至关重要。这项研究的可能结果将是对唾液Lipocalin序列的结构确定。有了这种结构，我们将能够更准确地确定导致Triabin和Nitoporin 2之间结构差异的序列要求，并了解该家族中的一些结构变异。了解蛋白质结构和功能的变化对于设计治疗靶点、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目标识别蛋白质的方法，该方法的序列介于已知差异性质的序列之间。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品的能力。

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.