The overlooked C‐terminal domain of <scp>IA3</scp> encodes a disordered concentration and salt sensor for inhibitory‐helix dynamics

Intrinsically disordered proteins (IDPs) often use charge-patterned polyampholyte regions to couple concentration and ionic strength to conformational changes, but the mechanisms by which such regions redistribute secondary-structure elements and modulate local dynamics remain incompletely understood. Here, we use the yeast vacuolar aspartyl protease inhibitor IA3 as a minimal model to dissect how a C-terminal polyampholyte controls the conformational ensemble and dynamics of a helix-forming N-terminal inhibitory segment. IA3 comprises a conserved N-terminal binding helix (residues 1-32), a mixed-charge linker (residues 33-49), and a near-neutral C-terminal polyampholyte (residues 50-68). We show by far-UV circular dichroism (CD) spectroscopy and BeStSel deconvolution that, at very low ionic strength, increasing IA3 concentration from 10 to 100 μM drives a reversible redistribution from helix/turn-enriched to antiparallel/coil-enriched ensembles, without visible phase separation. Raising salt to moderate levels recapitulates these high-concentration spectra at low micromolar IA3, indicating that electrostatic screening effectively reduces the interaction-driven "effective concentration" of chains. Single-site charge substitutions in the C-terminal region (D46K, E68N) reproduce the high-concentration ensemble at 10 μM, whereas other mutations (N52E, K61D, L60K, K24L) preserve the low-concentration, helix/turn-rich state. Site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) at position 9 within the inhibitory helix reveals that conditions favoring antiparallel/other-rich ensembles increase local mobility, whereas helix/turn-enriched states exhibit more restricted motion. These results establish IA3 as a minimal system in which a C-terminal polyampholyte functions as a tunable electrostatic sensor, coupling charge patterning, concentration, and ionic strength to secondary-structure redistribution and N-terminal helix dynamics. The findings define sequence-encoded electrostatic rules that may generalize to other polyampholyte-containing IDPs and suggest how IA3 couples its inhibitory activity to cellular ionic conditions.