Abstract
Selective proton transport through proteins is essential for forming and using proton gradients in cells. Protons are conducted along hydrogen-bonded ‘wires’ of water molecules and polar side chains, which, somewhat surprisingly, are often interrupted by dry apolar stretches in the conduction pathways, inferred from static protein structures. Here we hypothesize that protons are conducted through such dry spots by forming transient water wires, often highly correlated with the presence of the excess protons in the water wire. To test this hypothesis, we performed molecular dynamics simulations to design transmembrane channels with stable water pockets interspersed by apolar segments capable of forming flickering water wires. The minimalist designed channels conduct protons at rates similar to viral proton channels, and they are at least 106-fold more selective for H+ over Na+. These studies inform the mechanisms of biological proton conduction and the principles for engineering proton-conductive materials.
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Acknowledgements
Diffraction data were collected at the GM/CA@APS and ALS BL 8.3.1. GM/CA@APS is supported by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006 and P30GM138396), and the Eiger 16M detector by the National Institutes of Health (NIH; S10 OD012289). We also acknowledge the Advanced Photon Source, supported by the US Department of Energy (DE; contract DE-AC02-06CH11357), and beamline 8.3.1 at the Advanced Light Source operated by the University of California at San Francisco with support from the NIH (R01 GM124149 and P30 GM124169), Plexxikon and the Integrated Diffraction Analysis Technologies programme (US Department of Energy Office of Biological and Environmental Research). The Advanced Light Source at Lawrence Berkeley National Laboratory is supported by DE (DE-AC02-05CH11231). H.T.K. was supported by the NIH (K99GM138753). W.F.D. was supported by the NIH (R35 GM122603), NSF (CHE 1709506) and the Air Force Office of Scientific Research (FA9550-19-1-0331). L.C.W. and G.A.V. were supported by the NIH (R01 GM053148). J.M.N. was supported by the NIH (5T32HL007731 and F32GM133085). J.L.T. was supported by the NIH (R35GM122603).
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H.T.K. designed the channels, ran flux measurements, crystallized and collected the X-ray diffraction data, and ran and analysed the classical MD simulations. L.C.W. ran the MS-RMD simulations. L.C.W. and G.A.V. analysed the data. M.M. ran classical MD simulations. J.L.T, J.M.N. and L.L. processed and refined the crystal structures. H.T.K. and W.F.D. analysed the experimental data. All authors contributed to the data analysis and writing of the paper.
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Extended data
Extended Data Fig. 1 Composite omit maps (2mFo-DFc) of designed proton channels.
Composite omit maps of the asymmetric unit for a, LQLL, b, LLQL, and one pentamer from the asymmetric unit for c, QQLL, and d, QLQL (shown only for the waters for clarity). All contours at σ = 1.0. Omit maps with simulated cartesian annealing were generated using Phenix, using methodology described in Hodel, et al.104.
Extended Data Fig. 2 Pulse diagram and water-edited 13 C spectra, water buildup curves of membrane-bound LQLL and LLLL peptides.
a, Pulse diagram of water-edited 13 C CP experiment. b, Representative water-edited 13 C spectra of I13 in LQLL and LLLL, measured with 225 ms and 49 ms 1H mixing. The relative intensities of the 49 ms spectrum to the 225 ms spectrum are higher for LQLL Ile13 than LLLL Ile13, especially for the sidechain Cγ2 and Cδ carbons. c, Site-resolved water buildup curves for Ile13 in LQLL and LLLL. For all 13C sites, LQLL shows a faster water buildup than LLLL, consistent with water molecules in the pore lumen due to the Gln10 PLS.
Extended Data Fig. 3 Overview of proton flux measurements.
a, Full schematic for proton flux measurement including CCCP step, which is included to check vesicle leakiness and confirm proton selectivity. b, Chemical structures of key components of vesicle assay. c, Calibration curves for HPTS at ~5 µM in 12 solutions of 50 mM K2SO4, 30 mM K2HPO4 at different pH values for two plate readers used in data collection process. Unless stated, all data collected with instrument that generated the blue calibration curve. Fits used for downstream data processing shown for each of the two instruments with adjusted R-squared values of 0.9866 and 0.9998 for the left and right curves, respectively. Data for n = 3 independent samples shown as mean values +/− SD.
Extended Data Fig. 4 All proton flux assay data for long kinetics runs.
Long kinetics runs of about 5 hours total for a, empty, b, LLLL, and c, LQLL vesicles. Dotted lines denote times in experiment when valinomycin and CCCP were added to the samples. Three samples of the different conditions were measured in triplicate. These long-time measurements reveal that the vesicles are not leaky to Na+, K+, or H+ and maintain their cargo and assembly over the entire course of the measurement. d, From the linear regression fits of the first 220 s following addition of valinomycin, all slopes (which give the initial rates (in M/s)) were used to calculate the mean and standard error. The one-way ANOVA analysis (with Dunn’s test) reveals that LLLL rates are not significantly different (p > 0.05, adjusted p = 0.4021) when compared to the control empty vesicles. LQLL rates, however, are statistically significant (p < 0.0001, p = 3.23E-5) when compared to the control empty vesicles using one-way ANOVA analysis with Dunn’s test. All data from n = 3 independent samples are shown as mean values +/− SD.
Extended Data Fig. 5 All proton flux assay data for QLLL vesicle samples.
Nine samples (each run in triplicate with shaded error bars shown) containing 1:500 peptide:lipid ratio; samples were run independently in the assay. a, pHin as a function of time throughout the measurement for each independent sample. b, Mean and standard deviation for data collection. c, Data prior to CCCP addition shows little change in pHin after addition of valinomycin. d, Fits for the initial 50 seconds following addition of valinomycin. From the linear regression fits, all slopes (which give the initial rates (in M/s)) were used to calculate mean and standard error presented in Fig. 6g and Supplementary Table 3.
Extended Data Fig. 6 All proton flux assay data for LLQL vesicle samples.
Eight samples (each run in triplicate with shaded error bars shown) containing 1:500 peptide:lipid ratio; samples were run independently in the assay. a, pHin as a function of time throughout the measurement for each independent sample. b, Mean and standard deviation for data collection. c, Data prior to CCCP addition shows notable change in pHin after addition of valinomycin. d, Fits for the initial 50 seconds following addition of valinomycin. From the linear regression fits, all slopes (which give the initial rates (in M/s)) were used to calculate mean and standard error presented in Fig. 6g and Supplementary Table 3.
Extended Data Fig. 7 All proton flux assay data for QLQL vesicle samples.
Seven samples (each run in triplicate with shaded error bars shown) containing 1:500 peptide:lipid ratio; samples were run independently in the assay. a, pHin as a function of time throughout the measurement for each independent sample. b, Mean and standard deviation for data collection. c, Data prior to CCCP addition shows notable change in pHin after addition of valinomycin. d, Fits for the initial 50 seconds following addition of valinomycin. From the linear regression fits, all slopes (which give the initial rates (in M/s)) were used to calculate mean and standard error presented in Fig. 6g and Supplementary Table 3.
Extended Data Fig. 8 Determining orientation of pentamers in vesicles.
a, HPLC trace of unreacted and reacted peptides following reaction with the highly polar, amine-reactive methyltetrazine 3-sulfo-N-hydroxysuccinimide ester (methyltetrazine sulfo-NHS, see Methods). The only amine-reactive groups are the N-terminus or the N-terminal lysine sidechain. Thus, only peptides in which N-terminus is exposed on the outside of the vesicle should react to the dye. b, HPLC traces of mixtures of different ratios of non-reacted and reacted peptides. c, Calibration curves for area under the curve for nonreacted and reacted peaks in the HPLC traces corresponding to the different mixtures in b. Data shown are for n = 2 independent experiments and shown as mean values +/− SD. d, Traces of three independent samples of LQLL pentamers from vesicles after reaction with methyltetrazine sulfo-NHS. e, Using the calibration curves in c, the area under the curve was determined for each sample. The data indicate that half the amines react, as expected from a random orientation of pentamers in the lipid vesicle.
Supplementary information
Supplementary Information
Materials and Methods, Supplementary Figs. 1–25, Tables 1–6, a description of Video 1, and complete references and notes.
Supplementary Video 1
Snapshots from MS-RMD simulations along the MFEP (Fig. 5) depict the proton (represented as a yellow hydronium in the video) inducing the formation of water wires in hydrophobic regions of the channel lumen. The proton moves along these transiently forming water wires to make its way down to a region just below the PLS, formed by the green Gln residues. Once near this site, the presence of the proton enables the formation of a secondary water wire through the longer hydrophobic region. This allows the proton to traverse the rest of the way across the channel.
Source data
Source Data Fig. 2
Unclipped gel.
Source Data Fig. 4
Statistical source data.
Source Data Fig. 6
All data for every panel in Fig. 6, with each panel in unique tabs.
Source Data Extended Data Fig. 2
Statistical source data.
Source Data Extended Data Fig. 4
H+ concentration (M) for first 220 s after the addition of valinomycin for long kinetics runs.
Source Data Extended Data Fig. 5
All data for QLLL.
Source Data Extended Data Fig. 6
All data for LLQL.
Source Data Extended Data Fig. 7
All data for QLQL.
Source Data Extended Data Fig. 8
Data for calibration curves.
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Kratochvil, H.T., Watkins, L.C., Mravic, M. et al. Transient water wires mediate selective proton transport in designed channel proteins. Nat. Chem. 15, 1012–1021 (2023). https://doi.org/10.1038/s41557-023-01210-4
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DOI: https://doi.org/10.1038/s41557-023-01210-4