Pmel17 fibrils serve as the structural scaffolding required for melanin deposition in human skin and eyes. Melanin is synthesized in melanosomes, organelles related to both endosomes and lysosomes, and stored in melanocytes, cells responsible for pigmentation. While the melanosome maturation process has been shown to involve four distinct stages that have been characterized in detail at the ultrastructural level by transmission electron microscopy (TEM), the molecular nature of the intralumenal Pmel17 fibrils during each of these stages is not known. Moreover, which polypeptide domain solely or partly constitutes the amyloid core of the Pmel17 filaments also remains to be defined. We have begun to study the repeat domain (RPT, residues 315-444), an essential luminal polypeptide region of Pmel17, as a model system. To mimic the changing acidic pH conditions of the maturing melanosome, we measured RPT amyloid formation kinetics as a function of solution pH. Since tryptophan emission is highly sensitive to solvent polarity, local conformational changes, and protein-protein interactions, we exploited the only intrinsic tryptophan (Trp423) as a site-specific fluorescent probe of amyloid structure and aggregation kinetics. We find that Trp423 is exquisitely sensitive to soluble and fibrillar RPT conformation with spectral properties (intensity and mean wavelength) exhibiting distinct temporal changes under the various solution conditions examined. Complementary techniques, TEM and circular dichroism spectroscopy, also are employed to characterize fibrillar ultrastructures and secondary structural content, respectively.
| Jiang, Zhiping; Lee, Jennifer C (2014) Lysophospholipid-containing membranes modulate the fibril formation of the repeat domain of a human functional amyloid, pmel17. J Mol Biol 426:4074-4086 |
| McGlinchey, Ryan P; Jiang, Zhiping; Lee, Jennifer C (2014) Molecular origin of pH-dependent fibril formation of a functional amyloid. Chembiochem 15:1569-72 |
| McGlinchey, Ryan P; Yap, Thai Leong; Lee, Jennifer C (2011) The yin and yang of amyloid: insights from ?-synuclein and repeat domain of Pmel17. Phys Chem Chem Phys 13:20066-75 |
| McGlinchey, Ryan P; Gruschus, James M; Nagy, Attila et al. (2011) Probing fibril dissolution of the repeat domain of a functional amyloid, Pmel17, on the microscopic and residue level. Biochemistry 50:10567-9 |
| Pfefferkorn, Candace M; McGlinchey, Ryan P; Lee, Jennifer C (2010) Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid, Pmel17. Proc Natl Acad Sci U S A 107:21447-52 |
