The human voltage-gated proton channel (hH v 1) is a dimer of voltage sensors containing highly selective proton permeation pathways in each monomer. These voltage sensors are also activated by other stimuli, including pH gradients, mechanical forces, and ligand binding. Aside from the voltage sensors, this membrane protein contains an N-terminal domain and a C-terminal coiled-coil domain formed between the monomers. Fluorescence spectroscopy is a powerful technique for interrogating protein structure, as it enables the measurement of changes in the local environment or molecular distances through Förster resonance energy transfer (FRET). Here, we incorporated the fluorescent noncanonical amino acid acridon-2-ylalanine (Acd) into the full-length hH v 1 through genetic code expansion (GCE). Guided by a dimer AlphaFold structural model, we selected positions in the different structural domains of hH v 1 to incorporate Acd. We expressed and purified these hH v 1-Acd proteins, and showed they are stable and functional proton channels. The site-specific Acd incorporation was confirmed by steady-state and time-resolved fluorescence measurements. We measured site-specific FRET efficiencies in the hH v 1-Acd proteins when using Acd as a FRET acceptor of the intrinsic Trp and Tyr residues, and demonstrated that the protein is folded correctly in detergent micelles. Finally, reversible FRET changes were produced in the presence of the cation Zn 2+ , demonstrating that this classical inhibitor of hH v 1 currents changed the protein conformation. As this cation is bound in the experimental structural models of this protein, our data suggest that these structures correspond to a Zn 2+ -bound state that does not necessarily represent the Apo, resting, or closed conformations. Supported by NIH 5R01EY010329 and 5R35GM148137 to W.N.Z., NIH 1R01EY037223 and 5R35GM145225 to S.E.G., and the Pew Charitable Trusts to E.M.C.
Carmona et al. (Sun,) studied this question.