Summary In response to environmental stress cells often generate pH signals that serve to protect vital cellular components and reprogram gene expression for survival. A major barrier to our understanding of this process has been the identification of signaling proteins that detect changes in intracellular pH. To identify candidate pH sensors we developed a computer algorithm that searches proteins for networks of proton-binding sidechains. This analysis indicates that Gα subunits, the principal transducers of G protein-coupled receptor signals, are pH sensors. Our structure-based calculations and biophysical investigations reveal that Gα subunits contain networks of pH-sensing sidechains buried between their Ras and helical domains. We show further that proton binding induces changes in conformation that promote Gα phosphorylation and suppress receptor-initiated signaling. Together, our computational, biophysical and cellular analyses reveal a new and unexpected function for G proteins as mediators of stress-response signaling.

Protein folding is governed by a variety of molecular forces including hydrophobic and ionic interactions. Less is known about the molecular determinants of protein stability. Here we used a recently developed computer algorithm (pHinder) to investigate the relationship between buried charge and thermostability. Our analysis revealed that charge networks in the protein core are generally smaller in thermophilic organisms as compared to mesophilic organisms. To experimentally test whether core network size influences protein thermostability, we purified 18 paralogous Ras superfamily GTPases from yeast and determined their melting temperatures (Tm, or temperature at which 50% of the protein is unfolded). This analysis revealed a wide range of Tm values (35–63 °C) that correlated significantly (R = 0.87) with core network size. These results suggest that thermostability depends in part on the arrangement of ionizable side chains within a protein core. An improved capacity to predict protein thermostability may be useful for selecting the best candidates for protein crystallography, the development of protein-based therapeutics, as well as for industrial enzyme applications.

Cellular functionalities are contingent upon the ability of proteins to maintain or transform their shape (conformation). The molecular forces that govern protein folding have long been known. These include hydrophobic interactions, hydrogen bonding, and disulfide bonds. However, how proteins maintain their conformation remains largely unanswered. In this study, twenty G proteins from Saccharomyces cerevisiae were cloned, purified from E. coli, and tested for conformational stability. The proteins vary in Tm from 35 to 60°C. This is paradoxical considering their highly analogous crystal structures and identical catalytic activity. We find that thermal stability correlates with percentage of Cys, Met, or Pro residues in the primary sequence. We also find that the stabilities group by their respective family members, with the following trend observed for least to most thermostable G proteins: Rho/Cdc42 < Ran < Large G‐proteins < Ras < Rab < Sar/Arf. Both these results suggest that thermal stability has a greater dependence on primary sequence than tertiary structure. Ultimately, a more detailed understanding of protein stability can help in engineering enzymes for new molecular therapeutics. Grant Funding Source: Supported by National Science Foundation REU Site Award 1156840 to UNC‐Chapel Hill

Coordinated changes in intracellular pH accompany a number of cellular processes and stresses. However, we have limited knowledge of the specific cellular components that are regulated by intracellular pH signals. Here we introduce a bioinformatics approach for identifying a rare class of proteins that use core networks of acidic and basic sidechains to sense small changes in pH. Using this approach we have identified candidate proton sensors that participate in several signaling networks, including G protein and MAP kinase pathways. Through detailed biochemical, biophysical, and biological studies we have confirmed that these signaling proteins behave like proton sensors both in vitro and in vivo. Our experiments indicate that these sensors respond to proton binding by adopting conformations that are susceptible to phosphorylation, and thereby modulate pathway output. Based on these findings, we propose that cells use protons as second messengers to help regulate signal transduction under conditions of stress and pH‐coupled cellular processes.