Title: Transient Water Wires Mediate Selective Proton Conduction in Designed Channel Proteins Authors: Huong T. Kratochvil 1* , Laura C. Watkins 2 , Marco Mravic 1 , Jessica L. Thomaston 1 , John M. Nicoludis 1 , Lijun Liu 3 , Gregory A. Voth 2 , William F. DeGrado 1* Affiliations: 5 1 Department of Pharmaceutical Chemistry, University of California-San Francisco, San Francisco, CA 94158 2 Department of Chemistry, Chicago Center for Theoretical Chemistry, Institute for Biophysical Dynamics, and James Franck Institute, The University of Chicago, Chicago, IL 60637 10 3 DLX Scientific, Lawrence, KS, USA *Authors to whom correspondences should be addressed: huong.kratochvil@ucsf.edu, william.degrado@ucsf.edu Abstract: Selective proton transport through proteins is essential for forming and utilizing 15 proton gradients in cells. Protons are conducted along hydrogen-bonded “wires” of water molecules and polar sidechains, which, somewhat surprisingly, are often interrupted by dry apolar stretches in the conduction pathways inferred from static protein structures. We hypothesize that protons are conducted through such dry spots by forming transient water wires. To test this hypothesis, we used molecular dynamics simulations to design 20 transmembrane channels with stable water pockets interspersed by apolar segments capable of forming flickering water wires. The minimalist designed channels conduct protons at rates similar to viral proton channels, and they are at least 10 6 -fold more . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 28, 2022. ; https://doi.org/10.1101/2022.03.28.485852 doi: bioRxiv preprint