Ligand-coupled conformational changes in a cyclic nucleotide-gated ion channel revealed by time-resolved transition metal ion FRET
- Department of Physiology and Biophysics, University of Washington, United States
Figures
Figure 1
SthK as a model protein for characterizing the allosteric regulation in CNBD channels.
(A) Representative macroscopic A208V-cfSthK currents in inside-out patches from bacterial spheroplasts in response to voltage steps shown at the top, in the absence of cyclic nucleotide (top black …
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Figure 1—source data 1
Excel data for representative electrophysiology traces (Figure 1A) and summary of cyclic nucleotide dose response (Figure 1B).
- https://cdn.elifesciences.org/articles/99854/elife-99854-fig1-data1-v1.xlsx
Figure 2
Expression, purification, and analysis of tetrameric SthKCterm.
(A) In-gel protein fluorescence showing selective Acd incorporation into SthKCterm in the absence and presence of S361Acd TAG site, Acd aminoacyl tRNA synthetase/tRNA (RS/tRNA), and unnatural amino …
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Figure 2—source data 1
Original uncropped protein gel image (Figure 2A).
- https://cdn.elifesciences.org/articles/99854/elife-99854-fig2-data1-v1.zip
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Figure 2—source data 2
Labeled cropped protein gel image (Figure 2A).
- https://cdn.elifesciences.org/articles/99854/elife-99854-fig2-data2-v1.zip
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Figure 2—source data 3
Excel data for size exclusion chromatography (Figure 2D) and mass photometry data (Figure 2E).
- https://cdn.elifesciences.org/articles/99854/elife-99854-fig2-data3-v1.xlsx
Figure 3 with 1 supplement
Steady-state tmFRET data from tetrameric SthKCterm.
(A) Left: structure of one subunit of SthKCterm-S361Acd with [Fe(phenM)3]2+ acceptor incorporated at V416C (adapted from PDB: 4D7T; Kesters et al., 2015). Middle: Time course of average fluorescence …
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Figure 3—source data 1
Excel data for time courses and dot plots of steady-state FRET efficiencies and dose response (Figure 3A–D).
- https://cdn.elifesciences.org/articles/99854/elife-99854-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Steady-state tmFRET of monomeric and tetrameric SthKCterm.
(A) SEC chromatogram showing separation of monomeric (empty triangle, blue area) and tetrameric protein (solid triangle, gray area) fractions for SthKCterm-I359Acd and SthKCterm-I359Acd-R417C (no WT …
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Figure 3—figure supplement 1—source data 1
Excel data for size exclusion chromatography traces, monomer and tetramer steady-state time courses, and FRET efficiency summaries (Figure 3—figure supplement 1A–D).
- https://cdn.elifesciences.org/articles/99854/elife-99854-fig3-figsupp1-data1-v1.xlsx
Figure 4
Distance distribution predictions with chiLife.
(A) Structure of resting state (left, PDB:7RSH) (Gao et al., 2022), and cAMP-bound active state (right, PDB:4D7T) (Kesters et al., 2015) with rotameric clouds predicted by chiLife for the labels …
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Figure 4—source data 1
Excel data for chiLife distance distributions (Figure 4B).
- https://cdn.elifesciences.org/articles/99854/elife-99854-fig4-data1-v1.xlsx
Figure 5 with 2 supplements
Lifetime measurements of SthKCterm-S361Acd-V416C with [Fe(phenM)3]2+ and [Ru(bpy)2phenM]2+.
Chemical structures of acceptors and legends for all plots shown on top. (A–B) Representative Weber plots of phase delay and modulation ratio for SthKCterm-S361Acd-V416C labeled with [Fe(phenM)3]2+ …
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Figure 5—source data 1
Excel data for Weber plots, spaghetti plots and summary of Gaussian fit scatter plots (Figure 5A–F).
- https://cdn.elifesciences.org/articles/99854/elife-99854-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Representative Weber plot of measured lifetimes of SthKCterm-S361Acd donor-only (grey), then in the presence of [Fe(phenM)3]2+ (black), and after the addition of 1.23 mM cAMP (red), showing no change in lifetimes in the absence of a cysteine residue.
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Figure 5—figure supplement 1—source data 1
Excel data for fluorescence lifetimes Weber plot.
- https://cdn.elifesciences.org/articles/99854/elife-99854-fig5-figsupp1-data1-v1.xlsx
Figure 5—figure supplement 2
Parameters used in the lifetimes fitting model for both the single and sum of two Gaussian approaches, shown in blue.
(A) Graph of donor-only fluorescence-lifetime decay with two exponential components with time constants (, (B) FRET efficiency (E) plot as a function of distance (r) between donor and acceptor …
Figure 6 with 2 supplements
Analysis of lifetime data with global fit model allowing sum of two Gaussian distance distributions.
(A) Representative phasor plot of measured lifetimes with [Fe(phenM)3]2+ acceptor, where markers on universal circle indicate single-exponential time constants (in nanoseconds). Data shown are …
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Figure 6—source data 1
Excel data for phasor plot coordinates, Weber plot, spaghetti plot and dot plot for summary of Gaussian fit parameters from global fits (Figure 6A–E).
- https://cdn.elifesciences.org/articles/99854/elife-99854-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Representative phasor plot of measured lifetimes of SthKCterm-S361Acd-V416C alone (donor-only, grey), in the presence of [Ru(bpy)2phenM]2+ (apo, black), in the presence of 1 µM cAMP (cyan) and presence of 1.23 mM cAMP (red).
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Figure 6—figure supplement 1—source data 1
Excel data for phasor plot coordinates.
- https://cdn.elifesciences.org/articles/99854/elife-99854-fig6-figsupp1-data1-v1.xlsx
Figure 6—figure supplement 2
Identifiability of parameters in the sum of two Gaussian distributions model, with global fitting [Fe(phenM)3]2+ and [Ru(bpy)2phenM]2+ data.
(A–B) Minimization of values for the Gaussian average distance () (A) and standard deviation () (B) for the resting (black) and active states (red). (C) Minimization of values for …
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Figure 6—figure supplement 2—source data 1
Excel data for surface graphs for different model parameters (Figure 6—figure supplement 2A–D).
- https://cdn.elifesciences.org/articles/99854/elife-99854-fig6-figsupp2-data1-v1.xlsx
Figure 7
Lifetime measurements in 150 mM KCl versus 500 mM KCl conditions.
(A) Normalized SEC traces of SthKCterm in 150 mM KCl (solid curve) and 500 mM KCl (dashed curve). (B) Representative Weber plot for [Fe(phenM)3]2+ lifetimes in 150 mM (open circles) and 500 mM KCl …
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Figure 7—source data 1
Excel data for size exclusion chromatography traces, Weber plot, and dot plots for summary of Gaussian fit parameters from global fits (Figure 7A–D).
- https://cdn.elifesciences.org/articles/99854/elife-99854-fig7-data1-v1.xlsx
Tables
Table 1
Calculated energetics of the four state model.
| Ligand condition | Ionic strength (mM KCl) | ∆G (kcal/mol) | ∆∆G (kcal/mol) |
|---|---|---|---|
| Apo | 150 | 1.14±0.03 | -- |
| 500 | 1.2±0.03 | -- | |
| cAMP | 150 | -- | -- |
| 500 | <–2.7±0 | <–3.9±0.03 | |
| cGMP | 150 | 0.67±0.03 | –0.47±0.04 |
| 500 | 0.38±0.02 | –0.82±0.04 |
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Energies are reported in mean values ± SEM.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | B-95.ΔA E. coli | Addgene | Bacterial strain #197933 | |
| Recombinant DNA reagent | pDule2-Mj Acd A9 (plasmid) | Addgene | Plasmid #197652 | |
| Recombinant DNA reagent | MBP-S361TAG -ctermSthK-TEV-TWS.pETM11 (plasmid) | Addgene | Plasmid #231098 | |
| Recombinant DNA reagent | MBP- S361TAG -V416C-ctermSthK-TEV-TWS.pETM11 (plasmid) | Addgene | Plasmid #231099 | |
| Recombinant DNA reagent | MBP-Q364TAG -ctermSthK-TEV-TWS.pETM11 (plasmid) | Addgene | Plasmid #231100 | |
| Recombinant DNA reagent | MBP- Q364TAG -R417C-ctermSthK-TEV-TWS.pETM11 (plasmid) | Addgene | Plasmid #231101 | |
| Software, algorithm | FDlifetime_17_Igor_procedures.ipf | Zagotta, 2021 | https://github.com/zagotta/FDlifetime_program |
