Effects of Lipids on the Conformation and Aggregation of the Repeat Domain of a Functional Amyloid, Pmel17

Abstract

Pmel17 is a structural protein involved in melanin synthesis and deposition that forms fibrous striations in melanosomes, acidic organelles where pigmentation occurs. It was recently shown that a fragment of Pmel17 named the repeat domain (RPT, residues 315-444) is responsible for fibril formation in vitro under mildly acidic conditions. (McGlinchey RP. et al., PNAS, 2009; Pfefferkorn CM. et al., PNAS, 2010; McGlinchey RP. et al., JBC, 2010) Moreover, at neutral pH these fibrils disassemble, supporting a highly reversible aggregation/disaggregation process that could be a way for melanosomes to recycle amyloid fibrils. Here, we investigate the conformation and aggregation state of the RPT domain in the presence of membrane mimics, since it is localized in a membranous organelle. Specifically, micelles formed from detergents like sodium dodecyl sulfate or from lipids such as lysolipid as well as phospholipid vesicles were examined. Along with circular dichroism spectroscopy, which reports on the formation of secondary structure, we exploited the sole intrinsic Trp fluorophore (W423) located at the C-terminal region as a site-specific probe of interaction. To determine the specificity of this interaction, we also produced and examined single Trp mutants at the N-terminal region. Because the melanosome is an acidic organelle, we also explored the pH dependence of interaction in detail. Finally, we carried out aggregation experiments in the presence of lipid monomers and micelles in determining their effects on fibril formation.