Predicting and Characterizing the Guiding Principles of pH Sensitive Proteins

Intracellular pH (pHi) dynamics regulates various cell processes including cytoskeletal remodeling, growth, and metabolism. While these pH dependent behaviors have been well-characterized, little is known about the pH-sensing proteins (pH sensors) involved in these processes. A few cytoplasmic pH sensing-proteins have been identified, most of which have a key histidine conferring the pH-driven activity. However, in distinct protein microenvironments, the R group of Arginine, Lysine, Glutamate, Aspartate, Tyrosine, and Cysteine can have upshifted or downshifted pKas enabling titration within the physiological pH range. The potential role of these non-histidine residues in driving pH-dependent activity of cytoplasmic proteins is comparatively unexplored, even though their tunable pKa values suggest they could function as biologically important pH sensors beyond histidine. There is therefore a need to expand our understanding of pH sensitive protein allostery beyond the histidine-centric model uncovering new types of molecular pH switches and highlighting their roles in cellular regulation. The work described in this thesis demonstrates that pH sensing can also emerge from networks of ionizable residues that act cooperatively to confer sensitivity within the physiological pH range. We developed a structure-based bioinformatics pipeline, integrated with PROPKA3, to identify such networks by analyzing the spatial clustering of ionizable residues across conformational ensembles. Applying this pipeline to diverse panel of signaling proteins, including phosphatases (SHP2), kinases (Src), transcription factors (STAT3/5) and small G-proteins (RAN), revealed residue clusters that mediate distinct pH-sensitive activities, which we validated using constant pH molecular dynamics simulations, biochemical assays, and live-cell analyses. Collectively, this body of work expands our understanding of cytosolic pH sensing by uncovering alternative mechanisms beyond the well-established histidine-based switches. Future work will expand on the identified transcription factors (STAT proteins) with careful characterization of the predicted molecular mechanisms involved in their pH regulation.<p></p>