Observation of pH-Dependent Residual Structure in the Pmel17 Repeat Domain and the Implication for Its Amyloid Formation.

The varying conformational states of amyloid-forming protein monomers can determine their fibrillation outcome. In this study, we utilize solution 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. After excision from the full-length protein, RPT can self-assemble into amyloid fibrils, functioning as a scaffold for melanin deposition. Here, we report possible conformational states of the short RPT (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 nonaggregating solution conditions. We observed significant chemical shift perturbations localized to residues near the C-terminus, demonstrating that the local chemical environment of the amyloid core region is highly sensitive to changes in pH. Next, we introduced cysteine point mutations for the covalent attachment of PRE ligands to sRPT to facilitate the observation of intramolecular interactions. We also utilized solvent PRE molecules with opposing charges to measure changes in the electrostatic potential of sRPT in different pH environments. These observed PRE effects offer insight into initial molecular events that might promote intermolecular interactions, which can trigger fibrillation. Taken together, our results show that sRPT monomers adopt a conformation inconsistent with a fully random coil at neutral pH and undergo conformational changes at lower pH values. These observations highlight regulatory mechanisms via organelle-associated pH conditions that can affect the fibrillation activity of proteins like RPT.