(147c) pH-Controlled Hierarchical Assembly of Ion-Responsive Repeat Proteins

Electrostatic interactions facilitate ion capture and self-assembly by ion-binding proteins, and these interactions can be controlled by changes in pH. An exemplary class of ion-binding proteins is presented by the repeats-in-toxin (RTX) family, which includes repetitive bacterial proteins that exhibit drastic structural changes upon binding to calcium ions. The repeat motif includes a negatively charged aspartic acid residue that drives binding to positively charged calcium ions. The adenylate cyclase toxin (CyaA)—key to the virulence of whooping cough—is an RTX protein that harnesses the calcium-driven structural change for rapid translocation from bacteria to host. CyaA consists of five RTX blocks, with calcium-driven folding initiated by the fifth block (RTX-v) before propagating through the remaining repeat region (RTX-iv through RTX-i). Inspired by the coordinated, hierarchical assembly of multiple RTX blocks, we explore ion binding and folding of RTX-v in acidic conditions to reveal new insights into charge-driven assembly.

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.