Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Paris E. Taylor - Louisiana State University
Co-Author(s): Nathaniel E. Gilbert, Department of Biological Sciences, Louisiana State University, Baton Rouge, LA; Marcia E. Newcomer, Department of Biological Sciences, Louisiana State University, Baton Rouge, LA
Heart disease is the No. 1 killer for all Americans. The main focus of my research is to aid in new targeted therapies that interrupt the formation of oxidized low-density lipoprotein particles that cause heart disease. A critical component in the development of atherosclerosis is transformation of macrophages to foam cells, a process involving the uptake of oxidized lipids carried by low-density lipoprotein LDL. Recent studies have revealed that 15-lipoxygenase-2 (15-LOX-2), the major lipoxygenase expressed in macrophages, is found in high abundance in atherosclerotic plaque and is induced by hypoxia and other inflammation factors. The enzyme has been linked to elevated levels of oxidized lipids and is able to directly oxidize polyunsaturated fatty acids esterified in phospholipids. This study is aimed toward the engineering of a mutant form of 15-LOX-2 that will serve as a robust framework for future structural studies. Unfortunately, the wild type enzyme is not suitable for these studies because it crystallizes in such a way that we can’t trap inhibitors in the active site. Mutations will be made that facilitate crystallization, but do not change the overall characteristics of the binding site. To achieve this goal, we removed a loop from the enzyme that limited the quality of the crystals that can be made with this protein. This mutant 15-LOX-2 LM was expressed in E. Coli Rosetta 2 (DE3) cells. Once the protein was expressed, a crystallization tray was set up using the Sitting Drop Crystallization method. I used a reservoir solution of 16-20% Dimethyl Sulfoxide (DMSO), 16-20% Jeffamine M-2070, and deionized water. Equal volumes of reservoir and protein were suspended above the reservoir in a hanging drop. Protein crystals begin to appear after three weeks. The plate crystals are around 50 µm with a width of 5 µm. We will continue to use this protocol to prepare crystals of 15-LOX-2 LM with different inhibitors so that crystal structures can be determined. These crystal structures can provide information for structure-based drug design of new inhibitors of this enzyme.Not Submitted
Funder Acknowledgement(s): Dr. Marcia E. Newcomer Research Group, Department of Biological Sciences, Louisiana State University LSU IMSD Program (NIH 2R25GM069743)
Faculty Advisor: Marcia E Newcomer, firstname.lastname@example.org
Role: My biggest contribution to the study was the protein crystals trays I set up using the Sitting Drop Crystallization Method. With the different amounts of Dimethyl Sulfoxide, Jeffamine M-2070 and deionized water used, I was able to form macromolecular protein crystals. The plate crystals are around 50 µm with a width of 5 µm . I also helped prepare the proteins for crystallization by assisting in expression and purification of the 15-LOX-2 LM.