Molecular origin of pH-dependent fibril formation of a functional amyloid

Area of Science:

• Biochemistry
• Structural Biology
• Melanogenesis Research

Background:

• Pmel17 protein forms functional amyloid fibrils critical for melanin deposition within melanosomes.
• These Pmel17 repeat domain (RPT) fibrils exhibit pH-dependent stability, forming at acidic pH (4.5-5.5) and dissolving at neutral or alkaline pH (≥6).

Purpose of the Study:

• To identify the specific glutamic acid (Glu) residues within the Pmel17 RPT responsible for the pH-dependent reversibility of amyloid fibril formation.
• To elucidate the role of individual Glu residues in modulating the kinetics and stability of Pmel17 amyloid fibrils.

Main Methods:

• Site-directed mutagenesis was used to create single, double, and quadruple mutants of Pmel17 RPT, replacing Glu residues with Alanine (Ala) or Glutamine (Gln).
• Aggregation properties of wild-type and mutant fibrils were analyzed using intrinsic Tryptophan fluorescence, circular dichroism (CD) spectroscopy, and transmission electron microscopy (TEM).

Main Results:

• Charge neutralization of specific Glu residues (E404, E422, E425, E430) within the putative amyloid-forming region altered aggregation kinetics.
• Mutation of E422, a single negatively charged residue, significantly shifted the pH dependence of fibril formation by one full pH unit.
• Mutations at E404, E425, or E430 had minimal impact on fibril aggregation and stability.

Conclusions:

• Protonation of the glutamic acid residue at position 422 (E422) is essential for initiating Pmel17 amyloid fibril formation.
• Other glutamic acid residues in the vicinity appear to play an allosteric role in stabilizing the formed amyloid fibrils.