pH-Dependent fibril maturation of a Pmel17 repeat domain isoform revealed by tryptophan fluorescence.
Biochim Biophys Acta Proteins Proteom
; 1867(10): 961-969, 2019 10.
Article
en En
| MEDLINE
| ID: mdl-30716507
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 (381W). 381W emission properties revealed changes of local environment polarity for sRPT fibrils formed at different pH. At pHâ¯4, fibrils formed rapidly with no lag phase. A high 381W intensity was observed with a slight blue shift (10â¯nm). These fibrils underwent further structural rearrangements at intermediate pH (5-6), mirroring that of melanosome maturation, which initiates at pHâ¯4 and increases to near neutral pH. In contrast, typical sigmoidal kinetics were observed at pHâ¯6 with slower rates and 381W exhibited quenched emission. Interestingly, biphasic kinetics were observed at pHâ¯5 in a protein concentration-dependent manner. A large 381W blue shift (23â¯nm) was measured, indicating a more hydrophobic environment for fibrils made at pHâ¯5. Consistent with 381W 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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Antígeno gp100 del Melanoma
/
Agregado de Proteínas
/
Fluorescencia
Límite:
Humans
Idioma:
En
Revista:
Biochim Biophys Acta Proteins Proteom
Año:
2019
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Países Bajos