Interrogating the structure and function of the human voltage-gated proton channel (hHv1) with a fluorescent noncanonical amino acid

Figures

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  Figure 1.. Human voltage-gated proton channel (hHv1) was labeled with the fluorescent noncanonical amino acid acridon-2-ylalanine (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 1 mM 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…

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  Figure 1—figure supplement 1.. AlphaFold dimer human voltage-gated proton channel (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).

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  Figure 2.. Acridon-2-ylalanine (Acd) was incorporated into 14 positions in the human voltage-gated proton channel (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 (red arrows). The shorter detected bands in the WB for some constructs are produced by translation termination. 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. Figure 2—source data 1.Original files for acridon-2-ylalanine (Acd) fluorescence and Western blot showed in Figure 2 and analyzed in Figure 2—figure supplement 1 indicating the relevant bands. Figure 2—source data 2.Original files for acridon-2-ylalanine (Acd) fluorescence and Western blot showed in Figure 2 and analyzed in Figure 2—figure supplement…

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  Figure 2—figure supplement 1.. The protein yield of human voltage-gated proton channel (hHv1)-acridon-2-ylalanine (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 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). Figure 2—figure supplement 1—source data 1.OD600 measured at the end of the cultures expressing human voltage-gated proton channel (hHv1)-acridon-2-ylalanine (Acd) analyzed in Figure 2—figure supplement 1.

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  Figure 2—figure supplement 2.. The protein yield of human voltage-gated proton channel (hHv1)-A18Acd was proportional to the concentration of acridon-2-ylalanine (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. Figure 2—figure supplement 2—source data 1.Original files for acridon-2-ylalanine (Acd) fluorescence and Western blot showed and analyzed in Figure 2—figure supplement 2 indicating the relevant bands. Figure 2—figure supplement 2—source data 2.Original files for acridon-2-ylalanine (Acd) fluorescence and Western blot showed and analyzed in Figure 2—figure supplement 2. Figure 2—figure supplement 2—source data 3.OD600 measured at the end of the cultures expressing hHv1-A18Acd analyzed in…

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  Figure 2—figure supplement 3.. The truncation of human voltage-gated proton channel (hHv1)-K125Acd was not decreased at higher concentrations of acridon-2-ylalanine (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. Figure 2—figure supplement 3—source data 1.Original files for acridon-2-ylalanine (Acd) fluorescence and Western blot showed and analyzed in Figure 2—figure supplement 3 indicating the relevant bands. Figure 2—figure supplement 3—source data 2.Original files for acridon-2-ylalanine (Acd) fluorescence and Western blot showed and analyzed in Figure 2—figure supplement 3. Figure 2—figure supplement 3—source data 3.OD600 measured at the end of the cultures expressing hHv1-K125Acd analyzed in…

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  Figure 3.. Purification and functional measurements of human voltage-gated proton channel (hHv1)-acridon-2-ylalanine (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 9-Amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching…

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  Figure 3—figure supplement 1.. SDS-PAGE of the human voltage-gated proton channel (hHv1) protein samples after purification. Coomassie-stained (top) and acridon-2-ylalanine (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. Figure 3—figure supplement 1—source data 1.Original files for acridon-2-ylalanine (Acd) fluorescence and Western blot showed in Figure 3—figure supplement 1 indicating the relevant bands. Figure 3—figure supplement 1—source data 2.Original files for acridon-2-ylalanine (Acd) fluorescence and Western blot showed in Figure 3—figure supplement 1.

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  Figure 3—figure supplement 2.. The main human voltage-gated proton channel (hHv1) peak in the size exclusion chromatogram corresponds to the dimer. (Left) The No TAG sample was purified from cells expressing the hHv1 without any amber stop codon in the presence of acridon-2-ylalanine (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. (Right) SEC-cleaned samples were injected at the indicated concentration. The elution volumes of the samples were similar, with a mean ± standard deviation of 11.64 ± 0.01 mL. Additionally, a sample was run in a denaturing buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, and 0.2% SDS (dashed lines; elution volume of 12.5 mL). The invariant elution volume at low concentrations and higher elution volume in denaturing buffer strongly suggests that the main peak on the hHv1 chromatograms corresponds to the dimer. The delay between the fluorescence and absorbance peaks is due to the physical separation of the detectors. Ex/Em = 280/350 nm. Figure 3—figure supplement 2—source data 1.Original data for chromatograms showed in Figure 3—figure…

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  Figure 3—figure supplement 3.. Fluorescence-detection size exclusion chromatography of human voltage-gated proton channel (hHv1)-acridon-2-ylalanine (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.

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  Figure 3—figure supplement 4.. The purified human voltage-gated proton channel (hHv1)-acridon-2-ylalanine (Acd) proteins were functional proton channels. Liposome proton flux assays of asolectin proteoliposomes containing the indicated hHv1-Acd protein. The proteoliposome samples produced 9-Amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching after the addition of valinomycin (180 s) in the presence of a potassium gradient. The protonophore carbonyl cyanide m-chlorophenyl hydrazone (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.

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  Figure 4.. Acridon-2-ylalanine (Acd) showed a low environmental sensitivity in human voltage-gated proton channel (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. IRF: Instrument Response Function. (C) Normalized fluorescence emission spectra of the hHv1-Acd proteins in Buffer-H2 (gray). 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 3. (D) Normalized fluorescence decay of the hHv1-Acd proteins in Buffer-H2 (gray). The Acd amino acid decays in selected solvents are also shown for reference. Q56Acd and C107Acd decays are shown in red and black, respectively. Figure 4—source data 1.Original data for the emission fluorescence spectra of acridon-2-ylalanine (Acd) in different solvents showed in Figure 4. Figure 4—source data 2.Original data for the lifetime of acridon-2-ylalanine (Acd) in different solvents showed in Figure 4.…