RUI: Understanding headgroup-acylated glycerophospholipid biosynthesis and function in Escherichia coli

该项目提出了一个综合的研究和教育计划，让本科生参与大肠杆菌中一类未充分研究的脂质（称为头基酰化甘油磷脂（GPLs））的结构，功能和生物合成的原始研究。脂质是活细胞的关键部分，与其他大分子如蛋白质一起，它们形成包围所有细胞的膜。脂质支持基本的细胞功能，如细胞分裂和维持包围和界定细胞的膜。两个主要的研究目标是：了解影响细胞中头基酰化GPL水平的两种蛋白质的功能特性，以及头基酰化GPL水平在细菌中的调节方式;以及这些脂质水平的变化对膜弯曲程度的影响。这项工作将为理解这些次要但重要的脂质的功能奠定基础，并有助于在未来的工作中解释所有细胞中脂质的复杂性。该项目的第三个目标是直接让代表性不足的学生参与原创性研究。一个名为“潜入研究”的创新项目将六名被录取的瓦萨学院学生在大一开始前带到校园，参加为期10周的瓦萨大学本科夏季研究院的4到5周。通过早期与同行和教师研究导师建立密切联系，来自代表性不足群体的学生，包括低社会经济地位和第一代大学生，将走上STEM成功之路（科学，技术，工程和数学）的职业生涯。在整个项目中，本科生将参与对结构的原创性研究，在大肠杆菌中，一类被称为头基酰化甘油磷脂（GPLs）的脂质的功能和生物合成研究不足。 脂质（包括头基酰化GPL等低丰度脂质）的生物合成会影响细胞分裂和维持细胞膜完整性等基本细胞功能。 确定两种酶PldB和At 1g 78690的底物特异性和酶促机制，已知这两种酶影响E.大肠杆菌中，这些酶的体外产物的仔细结构测定，将提供有价值的见解的基础上，每一种酶的独特的催化机制。 参与此计画的学生将准备脂质基质及蛋白质萃取物，进行体外酵素分析，纯化酵素及建构必要的大肠杆菌。大肠杆菌菌株。 此外，他们将采用现代核磁共振和质谱技术来表征脂质结构。 了解改变头基酰化GPL水平在体内的影响将产生深入了解头基酰化GPL如何影响细胞分裂和维持膜的完整性。 旨在了解细胞如何调节头基酰化GPL水平以及这些脂质如何影响膜曲率的实验将使本科研究人员接触遗传学和细胞生物学技术，包括诱变和共聚焦显微镜。 这项工作将深入了解这类研究不足的脂质的生物合成和功能，这些脂质与它们在重要细胞过程中的新兴作用有关。 这个研究项目，这将有助于解释脂质在所有细胞的复杂性影响脂质生物化学领域，将积极影响参与本科生在瓦萨学院通过扩大他们的机会，从事大量的研究在脂质生物化学。 这项研究的每一个方面都将直接涉及本科生研究人员。他们将在科学会议上展示他们的工作作为海报和口头演讲，并在同行评审的期刊上发表，使这些学生走上在STEM相关领域成功职业生涯的道路。

This project presents an integrated research and education program to engage undergraduate students in original research on the structure, function and biosynthesis of a class of understudied lipids called headgroup-acylated glycerophospholipids (GPLs) in the bacterium Escherichia coli. Lipids are a key part of living cells and together with other macromolecules such as proteins, they form the membrane that surrounds all cells. Lipids support essential cell functions such as cell division and maintaining the membrane that surrounds and delimits the cell. The two main research objectives are: to understand the functional properties of two proteins that affect headgroup-acylated GPL levels in the cell as well as how levels of headgroup-acylated GPL are regulated in the bacterium; and the impact of changing levels of these lipids on how much the membrane can bend. This work will lay a basis for understanding the function of these minor, yet important, lipids and help, in future work, to explain the complexity of lipids in all cells. A third objective of this project is directly to engage underrepresented students in original research. An innovative program called Diving into Research brings six admitted Vassar College students to campus before the beginning of their freshman year to participate for 4 to 5 weeks in Vassar's 10-week Undergraduate Research Summer Institute. By establishing strong connections with peer and faculty research mentors early on, students from underrepresented groups, including low socioeconomic and first-generation college goers, are placed on a path to success in STEM (Science, Technology, Engineering and Mathematics)careers.Throughout this project, undergraduate students will be involved in original research on the structure, function and biosynthesis of a class of understudied lipids called headgroup-acylated glycerophospholipids (GPLs) in the bacterium Escherichia coli. The biosynthesis of lipids, including low abundance lipids such as headgroup-acylated GPLs, affects essential cell functions such as cell division and maintaining cell membrane integrity. Determining the substrate specificity and enzymatic mechanism of two enzymes, PldB and At1g78690, known to impact headgroup-acylated GPL levels in E. coli, and the careful structural determination of these enzymes in vitro products, will provide valuable insights into the basis of each enzyme's unique catalytic mechanisms. Students involved in this project will prepare lipid substrates and protein extracts, perform in vitro enzyme assays, purify enzymes and construct the necessary E. coli strains. In addition, they will employ modern techniques of nuclear magnetic resonance and mass spectrometry to characterize lipid structure. Understanding the impact of altering headgroup-acylated GPL levels in vivo will yield insights into how headgroup-acylated GPLs affect cell division and the maintenance of membrane integrity. Experiments designed to understand how cells regulate headgroup-acylated GPL levels and how those lipids impact membrane curvature will expose undergraduate researchers to techniques in genetics and cell biology, including mutagenesis and confocal microscopy. This work will yield insight into the biosynthesis and function of this under-investigated class of lipids related to their emerging roles in important cellular processes. This research project, which will influence the field of lipid biochemistry by helping to explain the complexity of lipids in all cells, will positively affect participating undergraduates at Vassar College by expanding their opportunity to engage in substantial research in lipid biochemistry. Every aspect of this research will directly involve undergraduate student researchers. They will present their work at scientific conferences as poster and oral presentations and publish in peer-reviewed journals, setting these students on a path to successful careers in STEM-related fields.