Structure of prion β-oligomers as determined by structural proteomics

Simple item page
dc.contributor.authorSerpa, Jason John
dc.contributor.supervisorBorchers, Christoph H.
dc.date.accessioned2017-09-07T14:32:11Z
dc.date.copyright2017en_US
dc.date.issued2017-09-07
dc.degree.departmentDepartment of Biochemistry and Microbiology
dc.degree.levelDoctor of Philosophy Ph.D.en_US
dc.description.abstractThe conversion of the native monomeric cellular prion protein (PrPC) into an aggregated pathological β-oligomeric (PrPβ) and an infectious form (PrPSc) is the central element in the development of prion diseases. The structure of the aggregates and the molecular mechanisms of the conformational change involved in this conversion are still unknown. My research hypothesis was that a specific structural rearrangement of normal PrPC monomers leads to the formation of new inter-subunit interaction interfaces in the prion aggregates, leading to aggregation. My approach was to use constraints obtained by structural proteomic methods to create a 3D model of urea-acid induced recombinant prion oligomers (PrPβ). My hypothesis was that this model would explain the mechanism of the conformational change involved in the conversion, the early formation of the β-structure nucleation site, and would describe the mode of assembly of the subunits within the oligomer. I applied a combination of limited proteolysis, surface modification, chemical crosslinking and hydrogen/deuterium exchange (HDX) with mass spectrometry for the differential characterization of the native and the urea-acid converted prion β-oligomer structures to get an insight into the mechanism of the conversion and aggregation. Using HDX, I detected a region of the protein in which backbone amides become more protected from exchange in PrPβ compared to PrPC. In order to obtain the inter-residue distance constraints to guide the assembly of the oligomer model, I then applied zero-length and short-range crosslinking to an equimolar mixture of 14N/15N-metabolically labeled β-oligomer thereby enabling the classification of the crosslinks as either intra-protein or inter-protein. Working with the Dokholyan group at the University of North Carolina at Chapel Hill, I was able to assemble a structure of the β-oligomer based on the combination of constraints obtained from all methods. By comparing the structures before and after the conversion, I was able to deduce the conformational change, that occurs during the conversion as the rearrangement and disassembly of the beta sheet 1– helix 1 – beta sheet 2 (β1-H1-β2) region from the helix 2 – helix 3 (H2-H3) core, forming new β-sheet nucleation site and resulting in the exposure of hydrophobic residues patches leading to formation of inter-protein contacts within aggregates.en_US
dc.description.embargo2018-06-14
dc.description.scholarlevelGraduateen_US
dc.identifier.bibliographicCitationPetrotchenko, E. V., Serpa, J. J., and Borchers, C. H. (2010) Analytical chemistry 82, 817-823en_US
dc.identifier.bibliographicCitationPetrotchenko, E. V., Serpa, J. J., and Borchers, C. H. (2011) Molecular & cellular proteomics : MCP 10, M110 001420en_US
dc.identifier.bibliographicCitationSerpa, J. J., Parker, C. E., Petrotchenko, E. V., Han, J., Pan, J. X., and Borchers, C. H. (2012) Eur J Mass Spectrom 18, 251-267en_US
dc.identifier.bibliographicCitationSerpa, J. J., Patterson, A. P., Pan, J., Han, J., Wishart, D. S., Petrotchenko, E. V., and Borchers, C. H. (2012) Journal of proteomicsen_US
dc.identifier.bibliographicCitationPetrotchenko, E. V., Serpa, J. J., Hardie, D. B., Berjanskii, M., Suriyamongkol, B. P., Wishart, D. S., and Borchers, C. H. (2012) Molecular & cellular proteomics : MCPen_US
dc.identifier.bibliographicCitationSerpa, J. J., Makepeace, K. A., Borchers, T. H., Wishart, D. S., Petrotchenko, E. V., and Borchers, C. H. (2013) Journal of proteomicsen_US
dc.identifier.bibliographicCitationPetrotchenko, E. V., Serpa, J. J., Makepeace, K. A., Brodie, N. I., and Borchers, C. H. (2014) Journal of proteomics 109, 104-110en_US
dc.identifier.bibliographicCitationPetrotchenko, E. V., Makepeace, K. A., Serpa, J. J., and Borchers, C. H. (2014) Methods Mol Biol 1156, 447-463en_US
dc.identifier.bibliographicCitationPetrotchenko, E. V., Serpa, J. J., Cabecinha, A. N., Lesperance, M., and Borchers, C. H. (2014) Journal of proteome researchen_US
dc.identifier.urihttp://hdl.handle.net/1828/8548
dc.languageEnglisheng
dc.language.isoenen_US
dc.rightsAvailable to the World Wide Weben_US
dc.subjectβ-oligomeren_US
dc.subjectprionen_US
dc.subjectPrPen_US
dc.subjectHDXen_US
dc.subjecthydrogen/deuterium exchangeen_US
dc.subjectcrosslinkingen_US
dc.subjectmass spectrometryen_US
dc.subjectlimited proteolysisen_US
dc.subjectsurface modificationen_US
dc.subjectprotein structureen_US
dc.subjectproteomicsen_US
dc.subjectstructural proteomicsen_US
dc.subjectprion structureen_US
dc.subjectdiscrete molecular dynamicsen_US
dc.subjectCL-DMDen_US
dc.subjectmisfoldingen_US
dc.subjectaggregationen_US
dc.titleStructure of prion β-oligomers as determined by structural proteomicsen_US
dc.typeThesisen_US

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
Serpa_Jason_PhD_2017.pdf
Size:
6.61 MB
Format:
Adobe Portable Document Format
Description:
Download
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
license.txt
Size:
1.71 KB
Format:
Item-specific license agreed upon to submission
Description:
Download

Collections

Electronic Theses and Dissertations (ETD)
 
University of Victoria Libraries

We acknowledge and respect the Lək̓ʷəŋən (Songhees and Esquimalt) Peoples on whose territory the university stands, and the Lək̓ʷəŋən and W̱SÁNEĆ Peoples whose historical relationships with the land continue to this day.