Date of Award
Doctor of Philosophy
Michael D. Best
David C. Baker, Francisco N. Barrera Olivares, Tessa R. Calhoun
Relationships between peripheral proteins and membrane-forming lipids are essential to proper cellular function and signalling. Particular lipids and lipid arrangements are known to recruit proteins to biomembrane surfaces to initiate conformational changes and enable proximity-promoted interactions with other proteins. However, globally identifying all unique protein-lipid interactions (PLI) and characterizing how they vary under changing circumstances is difficult to accomplish with traditional analytical methods, especially in vivo. In this work, we aim to solve this problem by synthesizing probe molecules that mimic specific classes of lipids, such as diacylglycerol (DAG), phosphatidic acid (PA), or phosphatidylserine (PS), and feature a photoaffinity tag for crosslinking of associated proteins and a bioconjugation handle to aid in characterization. We present the syntheses of clickable lipid photoaffinity probes that exhibit reduced synthetic modification from native lipid structures compared to past designs, and we demonstrate their efficacy in labeling proteins in cell lysates and in live cells as analyzed by SDS-PAGE and proteomic LC-MS.Additionally, we present metabolic probes for infiltrating and labeling lipid biosynthetic products in yeast. Metabolic labeling methods related to this aim explore novel ways to incorporate synthetic bioconjugation handles onto biomolecules using the cell's own enzymatic machinery. Labeled compounds can then be derivitized with a fluorescent dye or other group to aid analysis, and then visualized by whole-cell microscopy and characterized by extract thin-layer chromatography or mass spectrometry. Here, we introduce a collection of glycerol-based fatty monoester and monoether probes modified with an azide tag for labeling lipids in yeast.
Carr, Adam Judson, "Synthesis and Application of Biomimetic Probes for Investigating Protein-Lipid Interactions and Lipid Metabolism. " PhD diss., University of Tennessee, 2018.
Available for download on Thursday, August 15, 2019