Engineered Channel Asymmetry Extends Hydrogen-Bonding Networks for Proton Conduction

The precise and selective transport of protons across cellular membranes relies on the dynamic formation and dissipation of hydrogen-bonding networks involving water molecules, protein sidechains, and backbone carbonyls. As in aqueous solution, protons are conducted over long distances along chains of hydrogen-bonded water molecules within narrow protein pores. To engineer proton-conductive pathways, therefore, we must explicitly account for the dynamic behavior of these networks. In previous work, we showed that incorporation of polar Gln residues into hydrophobic pores drives formation of transient, single-file water wires that enable proton-selective transport. Here, we sought to enhance conduction by introducing targeted Ile-to-Ser substitutions to extend connectivity across the pore. We find that the position of Ser relative to Gln modulates sidechain dynamics and, in turn, channel hydration. Although increased polarity reduces hydrophobic length and enhances hydration, these effects alone do not explain the observed conduction rates. Instead, asymmetry in the arrangement and dynamics of polar sidechains emerges as a key determinant of proton conductivity. Together, these results demonstrate that proton conduction is governed not only by pore polarity and hydration, but also by the dynamic and asymmetric organization of hydrogen-bonding networks. This work establishes design principles for engineering proton-selective channels and reveals how asymmetry enables efficient proton transport across biological membranes. Significance StatementProton transport, central to many biocatalytic and bioenergetic processes, requires exceptional selectivity, yet the governing principles remain elusive and difficult to disentangle in natural systems. Designed minimalist proton channels offer new avenues to isolate and test features hypothesized to influence proton conduction. Here, we engineered new-to-nature proton channels with increasingly polar vestibules to establish how control of the hydrogen-bonding network tunes proton conductivity. We found that the effective hydrophobic length, pore polarity, and pore hydration do not contribute significantly to proton conduction rates. Instead, we show that asymmetric sidechain dynamics are key to extending the hydrogen-bonding networks necessary for rapid proton translocation. Our results reveal new tunable parameters that must be considered in the design of proton-selective systems.