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

生活在超过75摄氏度和pH值3的温度下，Sulfolobus sp .及其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，对硫虫蛋白和硫虫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。本项目的目的是研究磺胺虫蛋白和磺胺虫梭形病毒（SSV）蛋白的结构和功能，并确定在宿主-病毒系统中潜在的蛋白-蛋白相互作用。特别感兴趣的蛋白质包括硫虫热休克蛋白（HSPs）及其复合物，它们被归类为II族伴侣蛋白。HSP配合物，如双环纳米罗塞体，由60kDa α、β和γ亚基组合而成。此外，值得关注的还有SSV结构蛋白VP1、VP2和VP3，它们是在病毒生命周期中被指定功能的四种蛋白中的三种。该项目的两个具体目标是：(1)表征在野生型和热刺蛋白敲除中，硫虫生长和梭状病毒产生对不同环境条件的影响；(2)构建克隆、表达和纯化病毒蛋白，在体外检测热刺蛋白-副蛋白的潜在相互作用，在体内鉴定宿主蛋白-病毒蛋白的潜在相互作用。除了这些特定的目的，电子显微镜技术将用于阐明实验条件下病毒结构的不规则性，x射线晶体学将用于进一步探索蛋白质结构。环境样本也将从地热区域采集，这些区域尚未进行磺胺类化合物的筛选，以确定新的磺胺类化合物和ssv，或验证来自以前未充分研究的栖息地的特征物种的存在。更广泛的影响在过去三年中，Salish Kootenai学院（Pablo， MT）、蒙大拿大学（Missoula， MT）、蒙大拿州立大学（Bozeman， MT）和波特兰州立大学（PSU）的科学教师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攻读科学高级学位的人数。这个项目将通过提供一个共同感兴趣的研究项目来加强这些合作。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。具体来说，每年最多有两名本科生将得到本科生助教奖学金的支持，并将在PI及其合作者的实验室之间自由流动，以学习特定机构专门提供的技术和使用仪器。此外，该项目旨在支持来自大学合作伙伴的一名研究生，他将参加部落学院的暑期实习，作为近同伴指导和学生交换计划的一部分。其目标是增加土著学生使用高端仪器的机会，同时鼓励小型少数民族服务机构与大型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以研究为基础的研究生科学学位课程，对参与的土著学生来说是一种愉快的经历。PI目前为来自平头印第安保留地社区和全国其他部落社区的高中生和本科生提供暑期体验；该项目将通过为从这些拓展和教育项目中脱颖而出的优秀学生提供持续的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.