RUI: Probing the Beta-amyloidogenic Structure and Function of Hemolysin A From Proteus Mirabilis

University Of Wisconsin-La Crosse, La Crosse WI

Investigators

Abstract

One of the most interesting questions in biology today is the process by which properly folded proteins convert into insoluble helix amyloid fibrils. A hallmark feature of amyloid fibril formation is that a template-assisted model in which the surface of a mis-folded protein activates the mis-folding of nearby folded proteins. Currently, two different types of protein surfaces have been used during template-assisted amyloid fibril formation: (1) a cross beta interface and (2) an extended fibril-like interface. Hemolysin A (HpmA), a hemolytic toxin from Proteus mirabilis, will be used as a model to investigate the role of surface activation during a template-assisted hemolysis assay. HpmA belongs to the energy independent two-partner secretion (TPS) pathway, exhibits template-assisted activity, harbors a helix rich monomeric structure and forms oligomers in cross-beta and extended fibril conformations. Therefore, it serves as a good model to investigate surface interactions with regard to protein folding. Specific goals of this research are to test the hypothesis that either the cross-beta or fibril-like oligomeric surfaces within HpmA are utilized during the template-assisted activation of hemolysis. A series of site-directed mutants will be engineered based upon data from the recently solved x-ray structure for a truncated form of HpmA. These mutants will be designed to disrupt either the cross beta or fibril-like surfaces. Once generated, each mutant form of HpmA will be analyzed for template-assisted activity within a hemolysis assay. These studies will provide new structural and functional information regarding the role of surface activation within TPS pathways, the most commonly used protein secretion pathway in nature. In many aspects, HpmA structurally and functionally mimics amyloid type proteins. Therefore, the results from these studies may assist the understanding of how protein surfaces are used during template-assisted amyloid fibril formation. Broader Impact: The most important impact of the project is that it will be conducted at a primarily undergraduate institution with involvement of undergraduate students as one of the primary missions. It will further enhance the undergraduate research culture at the University of Wisconsin-La Crosse. Undergraduate students involved in the project will gain valuable research skills, learn new techniques, and collect and present their results both locally and nationally. In addition, the project has a K-12 outreach component in which a high school science educator will become familiar with the research project, develop new techniques and integrate these into her classroom. The high school students will benefit by applying these learned techniques toward an actual research project, which should strengthen their interest in science.