Acidic conditions probe the influence of residue protonation on calcium binding and the resulting conformational changes of RTX-v. We use circular dichroism to observe changes in secondary structure, specifically a transition from a random coil in the absence of calcium to a β-roll structure in the presence of calcium. This calcium-driven formation of a β-roll structure exposes hydrophobic residues, which lead to RTX-v precipitation and increased solution turbidity in neutral pH environments. In acidic conditions, RTX-v adopts a compact conformation—regardless of calcium concentration. The loss of a calcium-driven structural change under acidic conditions highlights the key role of electrostatic interactions in calcium-responsive folding. Additionally, the acid-dependent structure does not lead to increased turbidity, in contrast to the behavior of ion-bound RTX-v. To further investigate the distinction between ion-bound and protonated RTX-v, we employ microfluidic modulation spectroscopy (MMS) to observe intra- and intermolecular interactions of RTX-v in solution. MMS identifies similar β characteristics between the two compact structures; increased intermolecular interactions in the protonated structure are attributed to reduced precipitation compared to the calcium-bound structure. Such differences in aggregation and solubility indicate that protonated and calcium-bound structures are distinct. This work improves understanding of the impact of pH-driven charge modulation on protein self-assembly and aggregation. Such insights into the role of pH and ionic conditions are key to guiding the hierarchical assembly of ion-responsive proteins, enabling future investigations of stimuli-responsive biomaterials, ion sequestration, and fibrillar assembly.