This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. PrPC is a membrane-associated protein believed to be involved in cellular adhesion and signaling. Furthermore, the protein also exists in an infectious form that is linked to diseases such as Bovine Spongiform Encephalopathy (""""""""mad cow disease"""""""") and Creutzfeldt?Jakob disease. This infectious isoform, termed PrPSc, is also known as a prion. PrPSc is capable of acting as an infectious agent through templating of the misfolded state onto PrPC. The precise conversion mechanism how PrPC is converted to PrPSc, and how subsequently amyloid fibrils are formed, are currently unknown. Several potential intermediates have been identified, including a mainly ?-sheeted oligomer and several polymers. Using a recombinantly expressed hamster PrP as a model system, it has been shown that some of these multimers can be separated by size exclusion chromatography (SEC). However, due to the tendency of PrP to rapidly aggregate, these different forms have yet to be characterized structurally. We propose to characterize these isoforms using solution x-ray scattering at bio-SAXS beamline BL4-2. We have developed a new protocol for connecting a HPLC size exclusion column (SEC) column directly to the BL4-2 scattering instrument, allowing rapid analysis of the protein fractions as they are eluted. We have already succeeded in characterizing the normal physiological form of recombinant PrP using this HPLC setup (preliminary results discussed below), and we now propose to refine our protocol to analyze the other isoforms and study initial oligomeric assembly intermediates leading to the formation of infectious amyloid fibers. Additionally we will also explore the effects of varying the protein buffer and the addition of small compounds on PrPC using the BL4.2 high-throughput facilities.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001209-32
Application #
8362408
Study Section
Special Emphasis Panel (ZRG1-BCMB-P (40))
Project Start
2011-03-01
Project End
2012-02-29
Budget Start
2011-03-01
Budget End
2012-02-29
Support Year
32
Fiscal Year
2011
Total Cost
$2,737
Indirect Cost
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Remesh, Soumya G; Andreatta, Massimo; Ying, Ge et al. (2017) Unconventional Peptide Presentation by Major Histocompatibility Complex (MHC) Class I Allele HLA-A*02:01: BREAKING CONFINEMENT. J Biol Chem 292:5262-5270
VanderLinden, Ryan T; Hemmis, Casey W; Yao, Tingting et al. (2017) Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism. J Biol Chem 292:9493-9504
Zhang, Haonan; Qiao, Anna; Yang, Dehua et al. (2017) Structure of the full-length glucagon class B G-protein-coupled receptor. Nature 546:259-264
Niedzialkowska, Ewa; Mruga?a, Beata; Rugor, Agnieszka et al. (2017) Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization. Protein Expr Purif 134:47-62
de Vries, Robert P; Tzarum, Netanel; Peng, Wenjie et al. (2017) A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors. EMBO Mol Med 9:1314-1325
Tolbert, William D; Gohain, Neelakshi; Alsahafi, Nirmin et al. (2017) Targeting the Late Stage of HIV-1 Entry for Antibody-Dependent Cellular Cytotoxicity: Structural Basis for Env Epitopes in the C11 Region. Structure 25:1719-1731.e4
Yoon, Chun Hong; DeMirci, Hasan; Sierra, Raymond G et al. (2017) Se-SAD serial femtosecond crystallography datasets from selenobiotinyl-streptavidin. Sci Data 4:170055
Warelow, Thomas P; Pushie, M Jake; Cotelesage, Julien J H et al. (2017) The active site structure and catalytic mechanism of arsenite oxidase. Sci Rep 7:1757
Tzarum, Netanel; de Vries, Robert P; Peng, Wenjie et al. (2017) The 150-Loop Restricts the Host Specificity of Human H10N8 Influenza Virus. Cell Rep 19:235-245
Hettle, Andrew; Fillo, Alexander; Abe, Kento et al. (2017) Properties of a family 56 carbohydrate-binding module and its role in the recognition and hydrolysis of ?-1,3-glucan. J Biol Chem 292:16955-16968

Showing the most recent 10 out of 581 publications