The Laboratory of Protein Conformation and Dynamics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Methods Mol Biol. 2023;2551:79-93. doi: 10.1007/978-1-0716-2597-2_7.
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.
Premelanosomal 蛋白 (Pmel17) 是一种人类功能性淀粉样蛋白，它通过作为黑色素聚合的支架来促进色素沉着。这发生在黑色素体中，Pmel17 首先被蛋白水解成较小的结构域，这些结构域负责纤维的形成。我们的工作表明，Pmel17 重复结构域（RPT，残基 315-444）在类似于黑色素体中发现的酸性条件下体外形成淀粉样纤维。从机制上讲，这是由酸性残基的质子化驱动的，导致电荷中和和随后的聚集。有趣的是，酸性残基的去质子化导致快速解聚，突出了迄今为止仅在功能性淀粉样蛋白中观察到的纤维形成和溶解的可逆机制。在本章中，我们描述了如何使用外源性硫黄素-T 和内源性色氨酸荧光分别监测 pH 依赖性 RPT 聚集和解聚。这些方法也可以更广泛地适应于研究其他淀粉样系统的可逆性，包括功能性和致病性的淀粉样系统。
