Biophysical Society Conference | Tahoe 2023

Proton Reactions: From Basic Science to Biomedical Applications

Wednesday Speaker Abstracts

CONSERVED HYDROGEN-BONDED MOTIFS AS HUBS OF STABILITY FOR HUMAN WATER CHANNELS Philip Drewniak 1 ; Peng Xiao 1 ; Dylan Dingwell 1 ; Vladimir Ladizhansky 1 ; Leonid S. Brown 1 ; 1 University of Guelph, Physics, GUELPH, ON, Canada Aquaporins (AQPs) are ubiquitous channels conducting water (and in some cases other small molecules) across cell membranes. AQPs possess a unique hourglass topology, with 6 full transmembrane helices and two unusual half-helices residing in the reentrant loops, which are crucial for conductance of water and exclusion of protons (via so-called NPA motifs and aromatic/arginine filter). Interestingly, AQPs show strong conservation of hydrogen-bonded motifs involving those reentrant loops and the extracellular loop C, across all kingdoms of life. We explored the role of these conserved motifs in human aquaporin 1 (hAQP1) by combining site-directed mutagenesis, temperature-dependent hydrogen/deuterium (H/D) exchange, FTIR spectroscopy, and solid-state NMR. Previously, we obtained extensive chemical shifts assignments on hAQP1 expressed and isotopically labeled in yeast and reconstituted into lipids, which allowed us to monitor H/D exchange site-specifically, as a function of time and temperature. These experiments identified a number of residues strongly protected from the exchange, which, along with their strong conservation in other AQPs, suggested their importance in maintaining the hAQP1 fold. We also found that these residues are part of a cluster on the extracellular side of hAQP1, which includes loop C, half-helix HE with its linker, and ends of helices H2 and H5. Thermally induced unfolding of this cluster precedes cooperative unfolding of the whole transmembrane domain. Thus, we produced a number of mutants of these protected and/or conserved residues and tested their structure and stability by attenuated total reflectance (ATR) FTIR spectroscopy coupled with H/D exchange at different temperatures. The FTIR results showed that several conserved hydrogen-bonded residues supporting proper orientation of loop C and half-helix HE are indeed crucial for maintaining hAQP1 structure and stability, stressing the importance of hydrogen-bonding interactions in membrane protein folding, stability, and functionality.

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