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

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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.