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pH-Dependent fibril maturation of a Pmel17 repeat domain isoform revealed by tryptophan fluorescence
| Content Provider | Scilit |
|---|---|
| Author | Dean, Dexter N. Lee, Jennifer C. |
| Copyright Year | 2019 |
| Description | Journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics The pre-melanosomal protein (Pmel17) aggregates within melanosomes to form functional amyloid fibrils that facilitate melanin polymerization. The repeat domain (RPT) of Pmel17 fibrillates under strict acidic melanosomal pH. Alternative splicing results in a shortened repeat domain (sRPT), which also forms amyloid fibrils. Here, we explored the effects of pH and protein concentration on sRPT aggregation by monitoring the intrinsic fluorescence of the sole tryptophan at position 381 $(^{381}$W).$ ^{381}$W emission properties revealed changes of local environment polarity for sRPT fibrils formed at different pH. At pH 4, fibrils formed rapidly with no lag phase. A high$ ^{381}$W intensity was observed with a slight blue shift (10 nm). These fibrils underwent further structural rearrangements at intermediate pH (5–6), mirroring that of melanosome maturation, which initiates at pH 4 and increases to near neutral pH. In contrast, typical sigmoidal kinetics were observed at pH 6 with slower rates and$ ^{381}$W exhibited quenched emission. Interestingly, biphasic kinetics were observed at pH 5 in a protein concentration-dependent manner. A large$ ^{381}$W blue shift (23 nm) was measured, indicating a more hydrophobic environment for fibrils made at pH 5. Consistent with$ ^{381}$W fluorescence, Raman spectroscopy revealed molecular level perturbations in sRPT fibrils that were not evident from circular dichroism, transmission electron microscopy, or limited proteolysis analysis. Finally, sRPT fibrils did not form at pH ≥7 and preformed fibrils rapidly disaggregated under these solution conditions. Collectively, this work yields mechanistic insights into pH-dependent sRPT aggregation in the context of melanosome maturation. |
| Related Links | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700743/pdf |
| Ending Page | 969 |
| Page Count | 9 |
| Starting Page | 961 |
| ISSN | 15709639 |
| e-ISSN | 18781454 |
| DOI | 10.1016/j.bbapap.2019.01.012 |
| Journal | Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics |
| Issue Number | 10 |
| Volume Number | 1867 |
| Language | English |
| Publisher | Elsevier BV |
| Publisher Date | 2019-02-02 |
| Access Restriction | Open |
| Subject Keyword | Journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics Biochemistry and Molecular Biology Functional Amyloid Transmission Electron Microscopy Circular Dichroism Raman Spectroscopy |
| Content Type | Text |
| Resource Type | Article |
| Subject | Analytical Chemistry Molecular Biology Biochemistry Biophysics |
