hHv1 was labeled with the fluorescent noncanonical amino acid Acd by genetic code expansion.

(A) Cartoon showing the components of the genetic code expansion system used to incorporate Acd via amber codon suppression in hHv1. (B) AlphaFold structural model of the dimer hHv1 colored by subunit. (C) AlphaFold structural model of a single hHv1 subunit colored by the predicted Local Distance Difference Test (pLDDT). Model confidence was represented by a gradient from red to blue, indicating low to high pLDDT. (D) In-gel fluorescence and Western blot to evaluate Acd incorporation. An Acd fluorescence band corresponding to the molecular weight of full-length hHv1 (red arrow) was observed in cellular extracts after separation by SDS-PAGE only when cells contained the MjA9-AcdRS/tRNA plasmid (RS) and were grown in the presence of 1mM Acd (top). The Western blot against the His-tag present at the N-terminus of hHv1 confirmed that the band corresponded to the recombinant protein (bottom). Note the hHv1 truncated product produced when the amber stop codon is replacing F247 in the absence of the aminoacyl-tRNA synthetase/tRNA plasmid and Acd.

Acd was incorporated into 14 positions in the hHv1 sequence.

(A) Cartoon showing the amino acids selected to be replaced by an amber stop codon in the hHv1 secondary structure to incorporate Acd (stars). (B) AlphaFold dimer hHv1 structural model with the amino acids selected to be replaced by Acd as cyan spheres. (C) Acd fluorescence gel (top) and Western blot (bottom) of cellular extracts after separation by SDS-PAGE showing the expression of the Acd-labeled hHv1 mutants. The No TAG lane contains an extract from cells grown under identical conditions (in the presence of Acd and the aminoacyl-tRNA synthetase/tRNA pair) and expressing hHv1 without an amber stop codon.

Purification and functional measurements of hHv1-Acd proteins.

(A) Coomassie-stained (top) and Acd fluorescence (bottom) gels after separation by SDS-PAGE showing representative samples of the membrane extracts after solubilization (left) and the final purified protein after immobilized metal affinity chromatography (right). (B) Protein yield of the hHv1 protein with Acd replacing the amino acid at the indicated position. Note that V187Acd and Q233Acd contained no detectable protein after purification. The red line indicates the protein yield obtained from hHv1 without an amber stop codon in the presence of Acd and the aminoacyl-tRNA synthetase/tRNA pair (No TAG). (C) Representative fluorescence-detection size-exclusion chromatograms of the purified hHv1 proteins with Acd incorporated at the indicated amino acid position. Insets: AlphaFold dimer models, with the amino acid replaced by Acd shown as cyan spheres. (D) Representative liposome proton flux assay of asolectin proteoliposomes containing the indicated hHv1 protein. All purified proteins produced ACMA fluorescence quenching after the addition of valinomycin (Val). The protonophore CCCP was added at the end of the experiment as a control. The No TAG sample corresponds to proteoliposomes containing hHv1 without an amber stop codon expressed in the presence of Acd and the aminoacyl-tRNA synthetase/tRNA pair.

Acd showed a low environmental sensitivity in hHv1.

(A) Normalized fluorescence emission spectra of the Acd amino acid dissolved in the indicated solvent (Excitation = 370 nm). EtAc: ethyl acetate, OctOH: 1-octanol, ButOH: 1-butanol, EtOH: ethanol. (B) Normalized fluorescence decay of the Acd amino acid dissolved in the indicated solvent. (C) Normalized fluorescence emission spectra of the hHv1-Acd proteins in Buffer-H2 (grey). The Acd amino acid spectra in selected solvents are also shown for reference. Q56Acd and C107Acd spectra are shown with broken lines. The emission maxima are listed in Table S3. (D) Normalized fluorescence decay of the hHv1-Acd proteins in Buffer-H2 (grey). The Acd amino acid decays in selected solvents are also shown for reference. Q56Acd and C107Acd decays are shown in red and black, respectively.

Spectral FRET analysis using Trp and Tyr donors and Acd as acceptor.

(A) Fluorescence emission spectra of the intrinsic fluorescent amino acids Trp (black) and Tyr (black, broken lines), along with the Acd absorption spectrum in Buffer-H2 (blue). (B) AlphaFold dimer hHv1 structural model with the amino acids colored according to (C) and the native tryptophan and tyrosine residues shown as yellow spheres. (C) Normalized fluorescence spectra of the purified hHv1-Acd proteins (Excitation = 280 nm). The spectra were normalized by the Trp/Tyr fluorescence. The colors correspond to the indicated hHv1-Acd protein, and the remaining spectra are shown as grey traces in the figure. The No TAG sample (black) contains purified hHv1 protein without any amber stop codon expressed in the presence of Acd and the aminoacyl-tRNA synthetase/tRNA pair. (D) Example of the spectral FRET analysis procedure. The fluorescence spectrum of hHv1-Y134Acd obtained when exciting at 280 nm (black, F280) minus the normalized spectrum at the same excitation wavelength of the No TAG sample (grey, F280,NoTAG) produced the Acd emission spectrum shown in red (F280 – F280,NoTAG). Ratio A values were calculated by dividing this spectrum by the hHv1-Y134Acd fluorescence spectrum obtained by direct excitation at 370 nm (F370). (E) Ratio A traces of the hHv1-Acd proteins colored according to (C). The ratio between the spectra of free Acd amino acid excited at 280 and 370 nm (Ratio A0) is shown as broken lines. (F) Mean Ratio A – Ratio A0 values (410-480 nm; N=4) as a function of the predicted FRET efficiencies from the AlphaFold hHv1 structural model, colored according to (C). The broken line is the best fit to the equation (Ratio A – Ratio A0) = m(Predicted FRET efficiency).

FRET between Trp/Tyr and Acd reports a conformational rearrangement in response to Zn2+.

(A) Normalized fluorescence emission spectra of hHv1-K169Acd (Excitation = 280 nm) in Buffer-H2 in the absence of Zn2+ (black, Apo), in the presence of 1 mM Zn2+ (red), or in the presence of 1 mM Zn2+ and 2 mM EDTA (blue). Spectra were normalized by the maximum intensity of the Acd emission spectrum of the same sample excited at 370 nm. (B) Spectral FRET analysis of hHv1-K169Acd in Buffer-H2 in the absence of Zn2+ (black, Apo), in the presence of 1 mM Zn2+ (red), or in the presence of 1 mM Zn2+ and 2 mM EDTA (blue). (C) Spectral FRET analysis of the hHv1-Acd proteins in Buffer-H2 in the absence (black, Apo) or presence of 1 mM Zn2+ (red). N=4, ***p<0.005, **p<0.01.

AlphaFold dimer hHv1 structural model.

(A) Extracellular view of the model, showing each subunit in a different color. (B) One subunit of the dimer showing the position of the arginine gating charges in S4 (blue), the selectivity filter (red), and the charge transfer center (yellow).

The protein yield of hHv1-Acd depended on the position of Acd incorporation.

(A) Optical density at 600 nm (OD600) of the final cultures expressing the hHv1 with Acd replacing the indicated amino acid. (B) Relative protein yield calculated by multiplying the OD600 and Western blot band intensity of cellular extracts from the final cultures expressing the hHv1 with Acd replacing the indicated amino acid. Values of normalized by the No TAG condition, which are cells expressing the hHv1 without any amber stop codon in the presence of Acd and the aminoacyl-tRNA synthetase/tRNA pair (red broken line).

The truncation of hHv1-K125Acd was not decreased at higher concentrations of Acd in the culture medium.

(A) Cellular extracts of cells co-transformed with the MjA9-AcdRS/tRNA pair plasmid and hHv1-K125TAG and grown with the indicated concentration of Acd in the culture medium were separated by SDS-PAGE to visualize the hHv1 band (red arrow) by Acd fluorescence (top) and Western blot against the N-terminus His-tag (bottom). (B) Optical density at 600 nm (OD600) of the cultures at the end of the expression. (C) Relative yield obtained by multiplying the Western blot band intensity by the OD600, normalized by the value obtained at 1 mM Acd.

SDS-PAGE of the hHv1 protein samples after purification.

Coomassie-stained (top) and Acd fluorescence (bottom) gels of the samples from the elution of the nickel resin column, separated by SDS-PAGE. Note that V187Acd and Q233Acd did not contain appreciable amounts of protein. The No TAG condition consisted of samples purified from cells expressing the hHv1 without any amber stop codon in the presence of Acd and the aminoacyl-tRNA synthetase/tRNA pair.

Size exclusion chromatogram of the No TAG sample.

The No TAG sample was purified from cells expressing the hHv1 without any amber stop codon in the presence of Acd and the aminoacyl-tRNA synthetase/tRNA pair. The sample eluted from the immobilized affinity chromatography column was concentrated and injected into the size exclusion chromatography column.

Fluorescence-detection size exclusion chromatography of hHv1- Acd proteins.

Chromatograms of the purified hHv1 proteins with Acd incorporated at the indicated amino acid position. The AlphaFold dimer model with the amino acid replaced by Acd as cyan spheres is included.

The purified hHv1-Acd proteins were functional proton channels.

Liposome proton flux assays of asolectin proteoliposomes containing the indicated hHv1-Acd protein. The proteoliposome samples produced ACMA fluorescence quenching after the addition of valinomycin (180 s) in the presence of a potassium gradient. The protonophore CCCP was added at the end of the experiment (600 s). The No TAG sample (red trace) contains proteoliposomes containing hHv1 without any amber stop codon expressed in the presence of Acd and the aminoacyl-tRNA synthetase/tRNA pair. Empty asolectin liposomes (black traces) showed slow ACMA fluorescence quenching.

Fluorescence emission spectra of hHv1-Acd.

Spectra of the indicated hHv1-Acd protein sample obtained when exciting at 280 nm (black) and 370 nm (blue). The isolated spectrum of Acd (red) was obtained by subtracting the black trace from the normalized spectrum of the No TAG sample excited at 280 nm (grey).

Fluorescence emission spectra of the Acd amino acid in solution.

Spectra of Acd in Buffer-H2 using an excitation wavelength of 370 nm (blue) or 280 nm (red).

Changes in the fluorescence emission spectra of hHv1-Acd in the presence of zinc.

Spectra of the indicated hHv1-Acd protein sample obtained when exciting at 280 nm in the Apo state (black) and in the presence of 1 mM Zn2+ (red). Spectra were normalized by the maximum intensity of the Acd emission spectrum of the same sample excited at 370 nm.

The protein yield of hHv1-A18Acd was proportional to the concentration of Acd in the culture medium.

(A) Cellular extracts of cells co-transformed with the MjA9-AcdRS/tRNA pair plasmid and hHv1-A18TAG and grown with the indicated concentration of Acd in the culture medium were separated by SDS-PAGE to visualize the hHv1 band (red arrow) by Acd fluorescence (top) and Western blot against the N-terminus His-tag (bottom). (B) Optical density at 600 nm (OD600) of the cultures at the end of the expression. (C) Relative yield obtained by multiplying the Western blot band intensity by the OD600, normalized by the value obtained at 1 mM Acd.

Size-exclusion chromatography elution volumes of the hHv1 proteins.

Elution volumes were measured as the location of the maximum value of absorbance at 280 nm in the chromatograms.

Emission fluorescence spectrum maximum of Acd in different solvents.

The wavelength corresponds to the location of the maximum intensity of the emission fluorescence spectrum of the free amino acid dissolved in the corresponding solvent. EtAc: ethyl acetate, OctOH: 1-octanol, ButOH: 1-butanol, EtOH: ethanol.

The emission fluorescence spectrum maximum of the hHv1-Acd proteins.

The wavelength corresponds to the location of the maximum intensity of the emission fluorescence spectrum of the samples.