Interrogating the structure and function of the human voltage-gated proton channel (hH_v1) with a fluorescent noncanonical amino acid.

Abstract

The human voltage-gated proton channel (hH_v1) is a dimer of voltage-sensor domains (VSDs) containing highly selective proton permeation pathways in each monomer. In addition to voltage, hH_v1 is regulated by other stimuli, including pH gradients, mechanical forces, and ligands, such as Zn²⁺. Aside from the VSDs, this membrane protein contains an N-terminal domain and a C-terminal coiled-coil domain (CC) formed between the monomers. To address the need for direct measurements of conformational rearrangements in hH_v1, we developed a Förster resonance energy transfer (FRET) approach to measuring the conformational rearrangements in full-length hH_v1 purified from <i>E. coli</i>. We used genetic code expansion (GCE) to generate a library of 14 full-length hH_v1 constructs, each incorporating the fluorescent noncanonical amino acid acridon-2-ylalanine (Acd) at a different site throughout the various structural domains. Following the expression and purification of these hH_v1-Acd proteins, we found that 12 sites yielded stable and functional proton-permeable channels. The fluorescence properties of Acd at each site showed small site-specific differences. Furthermore, we measured site-specific FRET efficiencies from tryptophan (Trp) and tyrosine (Tyr) to Acd in the hH_v1-Acd proteins and found results consistent with correct folding in detergent micelles. Finally, the addition of Zn²⁺ produced reversible changes in FRET, with affected residues clustered on the intracellular side of the channel.