RIG: Archaeal HSP Complexes and Viral Proteins: A Structural Biology and Biophysics Study

生活在超过75摄氏度和pH 3的温度下，硫化叶菌属。它们携带的病毒为研究极端微生物提供了一个很好的实验系统。 然而，硫化叶菌蛋白和硫化叶菌病毒蛋白的研究仍处于起步阶段，对蛋白质功能和极端微生物多样性的进化机制知之甚少。 本项目的目标是研究硫化叶菌蛋白和硫化叶菌梭形病毒（SSV）蛋白的结构和功能，以及确定在这种宿主-病毒系统中潜在的蛋白质-蛋白质相互作用。特别感兴趣的蛋白质包括硫化叶菌热休克蛋白（HSP）及其复合物，其被分类为II组伴侣蛋白。 HSP复合物，如双环纳米Rosettasome，由60 kDa α、β和γ亚基的组合组成。 此外，感兴趣的是SSV结构蛋白VP 1、VP 2和VP 3，它们是在病毒生命周期中被指定功能的仅有的四种蛋白质中的三种。 本项目的两个具体目标是：（1）表征硫化叶菌生长和梭状病毒生产在野生型和HSP敲除作为不同环境条件的函数;（2）构建克隆，表达和纯化病毒蛋白，以测试潜在的HSP-VP体外相互作用，并确定潜在的体内宿主蛋白-病毒蛋白相互作用。 除了这些特定的目标，在电子显微镜技术将被用来阐明在实验条件下的病毒结构的不规则性和X射线晶体学将被用来进一步探索蛋白质结构。 还将从尚未进行硫化叶菌筛查的地热区采集环境样本，以努力确定新的硫化叶菌和SSVs，或验证以前未充分研究的栖息地中是否存在特征物种。（巴勃罗，蒙大拿州），蒙大拿大学蒙大拿州州立大学（博兹曼，MT），和波特兰州立大学（PSU）在过去的三年里，我们开展了重要的教学和研究合作，目标是增加美国原住民学生的数量攻读科学方面的高级学位 该项目将通过提供关于共同感兴趣的主题的研究计划来加强这些合作。 来自PI实验室的美国原住民本科生将参与与该项目相关的校内和校外研究活动。 具体而言，每年最多两名本科生将获得本科生助学金的支持，并将在PI及其合作者的实验室之间自由移动，以学习技术并使用特定机构专门提供的仪器。 此外，该项目旨在支持一名来自大学合作伙伴的研究生，他们将参加部落学院的暑期实习，作为近同伴指导和学生交流计划的一部分。 我们的目标是增加高端仪器的本地学生的可访问性，同时鼓励较小的少数民族服务机构和较大的大学合作伙伴之间的跨文化交流，以便从本科科学学位课程过渡到基于研究的研究生科学学位课程对于参与的本地学生来说是一种愉快的体验。 PI目前为来自平头印第安人保留地社区和其他部落社区的高中生和本科生提供暑期体验;该项目将通过为这些推广和教育计划中出现的特殊学生提供持续支持来补充正在进行的活动。

Living at temperatures that exceed 75 degrees C and pH 3, Sulfolobus spp. and the viruses they harbor provide a good experimental system to study extremophiles. Nevertheless, studies of Sulfolobus proteins and Sulfolobus virus proteins are still in their infancy and little is known about protein function and the evolutionary mechanisms underlying extremophile diversity. The goal of this project is to investigate the structure and function of Sulfolobus proteins and Sulfolobus Spindle Virus (SSV) proteins as well as to identify potential protein-protein interactions in this host-virus system. Proteins of particular interest include the Sulfolobus Heat-Shock Proteins (HSPs) and their complexes, which are classified as group II chaperonins. HSP complexes, such as the double-ring nanomeric rosettasome are composed of combinations of 60kDa alpha, beta, and gamma subunits. Also, of interest are the SSV structural proteins VP1, VP2, and VP3, which are three of only four proteins that have been assigned a function in the viral life cycle. Two specific aims of this project are to: (1) characterize Sulfolobus growth and fusellovirus production in wild-type and HSP-knockouts as a function of different environmental conditions; (2) construct clones, express, and purify viral proteins to test potential HSP-VP interactions in vitro and identify potential in vivo host protein-viral protein interactions. In addition to these specific aims, techniques in electron microscopy will be employed to elucidate irregularities in viral structure under experimental conditions and x-ray crystallography will be employed to further explore protein structure. Environmental samples will also be taken from geothermal regions that have yet to be screened for Sulfolobus in an effort to either identify novel Sulfolobales and SSVs or to verify the presence of characterized species from previously understudied habitats.Broader ImpactsScience faculty at Salish Kootenai College (Pablo, MT), The University of Montana (Missoula, MT), Montana State University (Bozeman, MT), and Portland State University (PSU) have developed significant teaching and research collaborations over the past three years with the goal of increasing the number of Native American students pursuing advanced degrees in the sciences. This project will serve to enhance these collaborations by providing a research program on a topic of common interest. Native American undergraduate students from the PI's laboratory will engage in both on-campus and off-campus research activities related to this project. Specifically, up to two undergraduates per year will be supported by undergraduate assistantships and will freely move between the laboratories of the PI and of his collaborators to learn techniques and use instrumentation that is specifically available at particular institutions. In addition, this project aims to support one graduate student from a university partner who will participate in a summer internship at the tribal college as part of a near-peer mentoring and student exchange program. The goal is to increase accessibility of high-end instrumentation to Native students while encouraging cross-cultural exchange between smaller minority-serving institutions and larger university partners so that transitioning from undergraduate science degree programs to research-based graduate science degree programs is a pleasant experience for participating Native students. The PI currently offers summer experiences for high school students and undergraduate students from the Flathead Indian Reservation community and other tribal communities across the Nations; this project will complement ongoing activities by providing continual support for exceptional students emerging from these outreach and educational programs.