CRYSTAL STRUCTURE OF THE PLANT FULL-LENGTH SSDNA BINDING PROTEIN STWHY2 IN FREE

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 植物对病原体的防御反应主要依赖于利用预先存在的物理屏障和诱导防御，这需要大量转录防御基因。Whirly蛋白家族是在研究这种防御基因的激活时被发现的。StWhy1被发现是马铃薯植物基因PR-10a的激活子，在防御反应中通过与单链构象中的启动子结合而被激活。该蛋白随后被结晶，并显示出新的四聚体涡旋糖样折叠。当我们试图进一步确定这个基因家族成员所使用的机制时，我们遇到了采用遗传学方法的困难。这些基因的各种敲除所显示的表型是罕见的和微妙的。由于我们怀疑这些蛋白的C-末端部分在Whirly功能中发挥着重要作用，我们希望获得该区域的结构数据。由于当时使用的StWhy1结构不是全长的，并且它有一个标记来代替C-末端，我们需要使用新的结构。StWhy1的一个类似物StWhy2显示出很大的结晶潜力，并希望利用这一特性获得自由形式和与单链DNA形成络合物的良好晶体。我们希望这些结构将有助于揭示当蛋白质自由或与DNA结合时C末端结构域的位置和潜在作用。这些知识将指导我们在实验室中设计新的实验，并引导我们更好地理解这个在植物抵御病原体方面发挥重要作用的蛋白质家族。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The plant defense response to pathogens relies primarily on the use of pre-existing physical barriers and inducible defenses, which require massive transcription of defense genes. The whirly protein family was discovered when studying the activation of such defense gene. StWhy1 was found as the activator of the potato plant gene PR-10a that is activated during defense response by binding to the promoter in its single stranded conformation. The protein was later crystallized and revealed novel tetramer whirligig-like¿ folding. As we tried to move forward in identifying the mechanism used by the members of this gene family, we have encountered difficulties taking the genetic approach. The phenotype shown by various Knockout of those genes are rare and subtle. Since we suspect the C-terminal part of those proteins to play big role in the Whirly function, we would like to obtain structural data of this region. Since the StWhy1 construct used then was not full length and it had a tag in place of C-terminal end, we need to use new construction. A paralogue of StWhy1, StWhy2, show a great potential for crystallization and hope to use this characteristic to obtain good crystals of the free form and the form in complex with ssDNA. We hope that those constructions will help reveal the location and potential role of the C-terminal domain when the protein is free or bound to DNA. This knowledge will guide our design of new experiments in the lab and lead us to better understanding of this protein family that plays an important role in plant defense against pathogens.