title: Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid, Pmel17.
abstract: Pmel17 is a functional amyloidogenic protein whose fibrils act as scaffolds for pigment deposition in human skin and eyes. We have used the repeat domain (RPT, residues 315-444), an essential luminal polypeptide region of Pmel17, as a model system to study conformational changes from soluble unstructured monomers to β-sheet-containing fibrils. Specifically, we report on the effects of solution pH (4 → 7) mimicking pH conditions of melanosomes, acidic organelles where Pmel17 fibrils are formed. Local, secondary, and fibril structure were monitored via intrinsic Trp fluorescence, circular dichroism spectroscopy, and transmission electron microscopy, respectively. We find that W423 is a highly sensitive probe of amyloid assembly with spectral features reflecting local conformational and fibril morphological changes. A critical pH regime (5 ± 0.5) was identified for fibril formation suggesting the involvement of at least three carboxylic acids in the structural rearrangement necessary for aggregation. Moreover, we demonstrate that RPT fibril morphology can be transformed directly by changing solution pH. Based on these results, we propose that intramelanosomal pH regulates Pmel17 amyloid formation and its subsequent dissolution in vivo.
authors.affiliation: Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8013, USA.
authors.forename: Candace M
authors.initials: CM
authors.lastname: Pfefferkorn
authors.forename: Ryan P
authors.initials: RP
authors.lastname: McGlinchey
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D020460:Melanosomes / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D014364:Tryptophan / Q000737:chemistry; D058951:gp100 Melanoma Antigen / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000682:Amyloid; C548637:PMEL protein, human; D058951:gp100 Melanoma Antigen; D014364:Tryptophan