The long-term goals of this application are to yield fundamental insight into the role of protein disorder in chaperone function and to better understand pH-modulated cellular processes. We focus on the bacterial chaperone HdeA, which is a member of a recently discovered class of stress-sensing proteins that become activated by the very conditions that cause their unfolding. As pathogenic enteric bacteria pass through the harshly acidic environment of the mammalian stomach, HdeA undergoes a pH-triggered transition from an inactive folded dimer to a chaperone-active partially unfolded monomer to prevent other unfolded proteins from acid-induced aggregation. Although a general mechanism for HdeA activity is understood, the structural details of the disordered monomeric active state have yet to be determined, preventing a complete understanding of its pH stress-sensing function. Through a multi-disciplinary interplay between simulation and experiment, we aim to 1) reconstruct the chaperone-active disordered ensemble as a function of pH and 2) characterize chaperone interaction with substrate. We will perform novel coarse-grained simulations to describe the long time-scale conformational dynamics of HdeA in different pH environments. From the coarse-grained ensemble, we will build all-atom models of the monomer and refine them against nuclear magnetic resonance (NMR) chemical shifts and residual dipolar couplings. Based on our experimentally refined ensemble of free chaperone, we will develop a coarse-grained model to simulate HdeA bound to an NMR-accessible substrate. The structure of the chaperone-substrate complex from simulation will be refined against fluorescence resonance energy transfer distances and NMR data. For the experimentally refined models of free and bound HdeA, we will perform all-atom constant pH molecular dynamics simulation to calculate the pKa's of all acid titratable residues. The pKa calculations will report on specific pH-modulated interactions (pH triggers) that contribute to monomer flexibility and substrate interaction. We will test our predicted pH triggers through biochemical mutational studies. Collectively, our efforts will allow us to fully uncover the pH-sensing mechanism of HdeA in bacterial pathogenicity and to gain insight into the manner in which chaperones interact with substrate.