In studying amyloid-forming proteins, it is imperative to determine the monomer conformational dynamics that precede fibrillation. This study utilizes NMR and the paramagnetic relaxation enhancement (PRE) effect to observe monomer properties of the repeat domain (RPT) from a human functional amyloid, premelanosomal protein (Pmel17). RPT is generated through Pmel17 post-translational processing during melanosome maturation, where melanin biogenesis occurs. At acidic melanosomal pH, RPT self-assembles into amyloid fibrils, functioning as a scaffold for melanin deposition. Here, we report dynamics of the short (sRPT) isoform, which has been demonstrated to be a fibrillation nucleator. NMR experiments were performed to determine conformational differences in sRPT by comparing aggregation-prone vs. non-aggregating solution conditions at pH ranges from 4 to 6, respectively. We observed significant chemical shift perturbations localized to residues near the important tryptophan, demonstrating that the local chemical environment of the amyloid core region is sensitive to changes in pH even in the monomer form. Next, we introduced several cysteine point mutations in order to covalently attach PRE ligands to sRPT for observation of intramolecular interactions. Long-range PRE effects indicate potential contacts to residues on opposite ends of sRPT. These PRE effects might be an indication of initial molecular events to facilitate intermolecular interactions, which can go on to trigger the fibrillation mechanism. These results also hint at a transient conformation that inhibits fibrillation. Thus, by raising or lowering solution pH, the relative population of this state would be modulated. Taken together, these results show that sRPT monomers adopt a conformation inconsistent with fully random coil at neutral pH and undergo conformational changes at lower pH values. These observations highlight the tight regulatory mechanisms that can affect fibrillation activity of proteins like RPT.
