Related Experiment Video
Updated: Aug 23, 2025
X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
Published on: May 13, 2020
Monitoring Kinetics of pH-Dependent Aggregation and Disaggregation of the Pmel17 Repeat Domain
Dexter N Dean1, Jennifer C Lee2
1The Laboratory of Protein Conformation and Dynamics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Abstract:
The premelanosomal protein (Pmel17) is a human functional amyloid that promotes pigmentation by serving as a scaffold for melanin polymerization. This occurs within the melanosome, where Pmel17 is first proteolyzed into smaller domain(s) that are responsible for fibril formation. Our work has shown that the Pmel17 repeat domain (RPT, residues 315-444) forms amyloid fibrils in vitro under acidic conditions similar to those found in melanosomes. Mechanistically, this is driven by the protonation of acidic residues, resulting in charge neutralization and subsequent aggregation. Interestingly, the deprotonation of acidic residues leads to rapid disaggregation, highlighting a reversible mechanism of fibril formation and dissolution thus far only observed for functional amyloid proteins. In this chapter, we describe how to monitor pH-dependent RPT aggregation and disaggregation using extrinsic thioflavin-T and intrinsic tryptophan fluorescence, respectively. These methods can also be adapted more broadly to investigate the reversibility of other amyloid systems, both functional and pathogenic.
Insights
The premelanosomal protein (Pmel17) repeat domain forms reversible amyloid fibrils in vitro. This pH-dependent process, crucial for pigmentation, involves protonation-driven aggregation and deprotonation-driven disaggregation.
Area of Science:
- Biochemistry
- Structural Biology
- Cell Biology
Background:
- Premelanosomal protein (Pmel17) is a functional amyloid essential for melanin polymerization and pigmentation.
- Pmel17 undergoes proteolysis within melanosomes, yielding domains that form amyloid fibrils.
- Functional amyloids exhibit unique properties distinct from pathogenic amyloids.
Purpose of the Study:
- To investigate the pH-dependent aggregation and disaggregation of the Pmel17 repeat domain (RPT).
- To elucidate the mechanism of reversible fibril formation and dissolution in a functional amyloid.
- To establish methods for monitoring these pH-dependent amyloid dynamics.
Main Methods:
- In vitro formation of Pmel17 RPT amyloid fibrils under acidic conditions.
- Monitoring aggregation using extrinsic thioflavin-T fluorescence.
- Monitoring disaggregation using intrinsic tryptophan fluorescence.
Main Results:
- Pmel17 RPT forms amyloid fibrils in vitro at acidic pH, mimicking melanosome conditions.
- Fibril formation is driven by protonation of acidic residues, leading to charge neutralization and aggregation.
- Deprotonation of acidic residues triggers rapid disaggregation, demonstrating reversibility.
Conclusions:
- The Pmel17 RPT exhibits a reversible pH-dependent mechanism for amyloid fibril formation and dissolution.
- This reversibility is a key characteristic of functional amyloids.
- The described fluorescence methods can be broadly applied to study other amyloid systems.
