pH MODULATION OF FIBRIL DISSOCIATION AND COPPER BINDING PROPERTIES OF THE PRION PROTEIN

<p>The cellular form of prion protein (PrP^C) is a cell-surface glycoprotein attached to lipid rafts via its glycosylphosphatidylinositol anchor. Conversion of PrP^C to its "scrapie" conformer (PrP^Sc, the fibrillar form) constitutes the key event of the etiology of prion diseases. Fibril dissociation is necessary for efficient conversion and continued propagation of the disease state. Recent studies have revealed that conversion occurs along the endocytic pathway. To better understand the dissociation process, we have investigated the effect of low pH on the stability of recombinant prion fibrils. We show that under conditions that mimic the endocytic environment, amyloid fibrils made from full-length recombinant prion protein dissociate both laterally and axially to form protofilaments. About 5% of the protofilaments are short enough to be considered soluble and contain ~100-300 monomers per structure; these also retain the biophysical characteristics of the filaments. We propose that protonation of His residues and charge repulsion in the N-terminal domain trigger fibril dissociation. Our data suggest that lysosomes and late endosomes are competent milieus for propagating the misfolded state not only by destabilizing the normal prion protein, but by accelerating fibril dissociation into smaller structures that may act as seeds for further fibril formation.</p> <p>PrP^C binds four Cu(II) in its octarepeat region and another at the fifth binding site. Previous work has demonstrated detailed structural information on copper binding to these sites at neutral pH. Both types of binding sites contain ionizable groups, thus the effect of pH on copper binding needs to be clarified. Moreover, much less attention has been devoted to understanding copper binding in PrP^Sc, which is more pathologically relevant. These two aspects are investigated here using isothermal titration calorimetry and X-band electron paramagnetic spectroscopy. Our results confirm that copper binding to both the octarepeats and the fifth binding site is pH-dependent. We show that both sites bind copper in the fibrillar form with coordination modes similar to their monomeric counterparts. However, the ratios of the different coordination modes have changed in the fibril, which might suggest changes in their affinities after conversion and have potential effects on the redox properties of fibrils.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Grantor dc:publisher

University of Montana

Year dc:date.available

2012

Author dc:creator

• Qi, Xu

• Amyloid fibrils
• Copper
• EPR
• Low-pH
• Prion protein
• protofilaments

Repository record dc:identifier

https://scholarworks.umt.edu/etd/745

OAI identifier oai:identifier

oai:scholarworks.umt.edu:etd-1764