X-ray crystal structures of PsaA (3ZK7; cluster A), MalE (1OMP; cluster B), OppA (3FTO; cluster C), OpuAC (3L6G; cluster F), SBD1 (4LA9; cluster F) and SBD2 (4KR5; cluster F) are all shown in the …
(A) Experimental strategy to study SBP conformational changes via FRET. Solution-based apparent FRET efficiency histograms of OpuAC(V360C/N423C) (B), PsaA(V76C/K237C) (C), MalE(T36C/S352C) (D), …
Apparent FRET efficiency histograms of Figure 2B–G.
Apparent FRET efficiency histograms of Figure 2—figure supplement 3.
Representative fluorescence trajectories (left) and apparent FRET efficiency histograms from all fluorescence trajectories (right) of MalE(T36C/S352C) (A), SBD2(T369C/S451) (B), OpuAC(V360C/N423C) (C…
(A) Representative fluorescence trajectories of OppA(A209C/S441C) at different peptide (RPPGFSFR) concentrations; donor (green) and acceptor (red) photon counts. The top panel shows the calculated …
Solution-based apparent FRET efficiency histogram of MalE(T36C/S352C) (A) and OppA(A209C/S441C) (B) in the absence and presence of different cognate substrates as indicated. The OppA substrates are …
Solution-based apparent FRET efficiency histogram of MalE(T36C/S352C), MalE(T36C/N205C) and MalE(K34C/R354C) in the absence and presence of different cognate substrates as indicated. Bars are the …
(A) Schematic of the experimental strategy to study the conformational dynamics of ligand-free SBPs. Representative fluorescence trajectories of OpuAC(V360C/N423C) (B), PsaA(V76C/K237C) (C), …
Donor and acceptor photon counts, apparent FRET efficiency and most probable state-trajectory of the Hidden Markov Model of the traces in Figure 3.
Representative fluorescence trajectories of OpuAC(V360C/N423C) (A), PsaA(V76C/K237C) (B), MalE(T36C/S352C) (C), SBD1(T159C/G87C) (D), OppA(A209C/S441C) (E) and SBD2(T369C/S451) (F) in the absence of …
Closing rate (A) and average lifetime of the closed conformation (B) for OppA, SBD1 and SBD2 in the absence of ligand and in the presence of different concentrations of unlabeled protein to scavenge …
(A) Time-dependent uptake [14C]-asparagine (5 μM), [14C]-glutamine (5 μM), [14C]-arginine (100 μM), [14C]-histidine (100 μM) and [3H]-lysine (100 μM) by GlnPQ in L. lactis GKW9000 complemented in …
Apparent FRET efficiency histograms of Figure 4D–H.
The mean apparent FRET change of SBD1 (top) and SBD2 (bottom) in the presence of 5 mM of the indicated amino acids relative to their absence; measurements were performed in 50 mM KPi, 50 mM KCl, pH …
Solution-based apparent FRET efficiency histograms of SBD1(T159C/G87C) (A and C) and SBD2(T369C/S451) (B) in the presence of different ligand concentrations as indicated. Bars are the data and the …
Solution-based apparent FRET efficiency histogram of PsaA(E74C/K237C) in the presence and absence of metals as indicated. Bars are the data and solid line a Gaussian fit. The 95% confidence interval …
Solution-based apparent FRET efficiency histograms of PsaA(V76C/K237C) in the presence of Mn2+ (A) or Zn2+ (B) and PsaA(D280N) in the presence of Zn2+ (C) upon addition of 10 mM EDTA and incubated …
(A) Mean lifetime of the ligand-bound conformations of MalE, obtained from all single-molecule fluorescence trajectories in the presence of different maltodextrins as indicated. Data corresponds to …
Lifetimes of the high FRET state of the data shown in Figure 6A and Figure 6—figure supplement 2.
Donor and acceptor photon counts, apparent FRET efficiency and most probable state-trajectory of the Hidden Markov Model of the traces in Figure 6B–G.
Lifetimes of the high FRET state of the data shown in Figure 6—figure supplement 3B.
(A) Surface-based apparent FRET efficiency histogram of MalE(T36C/S352C) in the presence of different maltodextrin substrates as indicated. From the probable state-trajectory of the Hidden Markov …
Dwell time histogram of the high FRET (closed ligand-bound conformation) as obtained from the most probable state-trajectory of the Hidden Markov Model (HMM) of all molecules per condition as shown …
(A) Representative fluorescence trajectories of MalE(T36C/S352C/A96W/I329W) in the presence of 10 nM maltose. Fluorescence trajectories: the top panel shows the calculated apparent FRET efficiency …
Schematic summarizing the plasticity of ligand binding and solute import via ABC importers. Intrinsic closing of an SBP is a rare event or absent in some SBPs (‘little intrinsic closing’). Ligands …
KD (µM) | |||||
---|---|---|---|---|---|
Protein* | Ligand | Freely-diffusing protein | Surface-tethered protein | KD WT protein¶ (µM) | |
OpuAC(V360C/N423C) | Glycine betaine | 3.4 ± 0.4† | 3.1‡ | 4–5 (Wolters et al., 2010) | |
OppA(A209C/S441C) | RPPGFSFR | 7.0 ± 1† | 14 ± 5# | 5 ± 3# | |
SBD2(T369C/S451) | Glutamine | 1.2 ± 0.2§ | 0.5‡ | 0.9 ± 0.1 (Gouridis et al., 2015) | |
SBD1(T159C/G87C) | Asparagine | 0.34 ± 0.03§ | 0.3‡ | 0.2 ± 0.0 (Gouridis et al., 2015) | |
MalE(T36C/S352C) | Maltose | 1.7 ± 0.3† | 2.2‡ | 1-2 (Hall et al., 1997a, Kim et al., 2013) | |
MalE(T36C/S352C) | Maltotriose | 0.6 ± 0.2† | 0.9‡ | 0.2-2 (Hall et al., 1997a, Kim et al., 2013) |
*. KD could not be determined reliably for labeled PsaA due to background metal contamination.
†. Population of the closed conformation in the presence of a ligand concentration was determined using solution-based smFRET. The for a one-binding site model. Data corresponds to mean ± s.d. of duplicate experiments with the same protein sample.
#. Figure 2—figure supplement 2
¶. The KD values of wildtype (WT) proteins are obtained from the indicated references.
Anisotropy | ||||
---|---|---|---|---|
Alexa555 | Alexa647 | Cy3B | Atto647N | |
Free dye | 0.25 | 0.20 | 0.08 | 0.08 |
OpuAC(V360C/N423C) | NA | NA | 0.17 | 0.11 |
OppA(A209C/S441C) | 0.25 | 0.19 | NA | NA |
SBD1(G87C/T159C) | 0.27 | 0.19 | NA | NA |
SBD2(T369C/S451) | 0.26 | 0.20 | NA | NA |
MalE(T36C/S352C) | 0.29 | 0.24 | NA | NA |
PsaA(V76C/K237C) | 0.28 | 0.22 | NA | NA |
NA: not applicable. Data correspond to mean (s.d. below < 0.01) of duplicate experiments, using the same labeled protein sample.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene(Escherichia coli) | MalE | NA | UniProt: P0AEX9 | |
Antibody | Mouse anti-his | Qiagen | RRID:AB_2714179 | (1:200) |
Strain, strain background (Streptococcus pneumoniae) | D39 | National Collection of Type Cultures | NCTC:7466 | Capsular serotype 2 |
Strain, strain background (Streptococcus pneumoniae) | D39 ∆psaA | This paper | Replacement of psaA with the Janus cassette (∆psaA::Janus) | |
Strain, strain background (Streptococcus pneumoniae) | D39 ∆czcD | This paper | Replacement of czcD with the Janus cassette (∆czcD::Janus) | |
Strain, strain background (Streptococcus pneumoniae) | D39 ΩpsaAD280N | This paper | Replacement of ∆psaA::Janus with psaA D280N (∆psaA::psaAD280N) | |
Strain, strain background (Streptococcus pneumoniae) | D39 ΩpsaAD280N∆czcD | This paper | Replacement of ∆psaA::Janus with psaA D280N; replacement of czcD with the Janus cassette (∆psaA::psaAD280N∆czcD::Janus) | |
Strain, strain background (Lactococcus lactis) | NZ9000 | NIZO food research | ||
Strain, strain background (Lactococcus lactis) | GKW9000 | DOI: 10.1038/ nsmb2929 | Lactococcus lactis NZ9000 with glnPQ gene deleted | |
Strain, strain background (Escherichia coli) | K12 | Other | Provided by Tassos Economou, KU Leuven | |
Strain, strain background (Escherichia coli) | BL 21 DE3 | Other | Provided by Tassos Economou, KU Leuven | |
Recombinant DNA reagent | pET20b | Merck | Cat#:69739–3 | |
Recombinant DNA reagent | pNZglnPQhis | DOI: 10.1047/ jbc.M500522200 | Expression plasmid for GlnPQ | |
Recombinant DNA reagent | SBD1-T159C/G87C | DOI: 10.1038/ nsmb2929 | Expression plasmid for SBD1(T159C/G87C) | |
Recombinant DNA reagent | SBD2-T369C/S451C | DOI: 10.1038/ nsmb2929 | Expression plasmid for SBD2(T369C/S451C) | |
Recombinant DNA reagent | pCAMcLIC01-PsaA | DOI: 10.1038/ nchembio.1382 | Expression plasmid for PsaA | |
Recombinant DNA reagent | pCAMcLIC01-PsaAD280N | DOI: 10.1038/ nchembio.1382 | Expression plasmid for PsaA(D280N) | |
Recombinant DNA reagent | pNZOpuCHis | DOI: 10.1093/ emboj/cdg581 | Expression plasmid for OpuAC | |
Recombinant DNA reagent | pNZcLIC-OppA | DOI: 10.1002/pro.97 | Expression plasmid for OppA | |
Recombinant DNA reagent | PsaA-V76C/K237C | This paper | Expression plasmid for PsaA(V76C/K237C) from the pCAMcLIC01-PsaA construct | |
Recombinant DNA reagent | PsaA-E74C/K237C | This paper | Expression plasmid for PsaA(E74C/K237C) from the pCAMcLIC01-PsaA construct | |
Recombinant DNA reagent | PsaA-D280N/V76C/K237C | This paper | Expression plasmid for PsaA(D280N/V76C/K237C) from the pCAMcLIC01-PsaAD280N construct | |
Recombinant DNA reagent | MalE-T36C/S352C | This paper | Progenitors: PCR, E. coli gDNA; pET20b vector | |
Recombinant DNA reagent | MalE-T36C/N205C | This paper | Progenitors: PCR, E. coli gDNA; pET20b vector | |
Recombinant DNA reagent | MalE-K34C/ R354C | This paper | Progenitors: PCR, E. coli gDNA; pET20b vector | |
Recombinant DNA reagent | MalE-T36C/S352C/ A96W/I329W | This paper | Progenitors: PCR, E. coli gDNA; pET20b vector | |
Recombinant DNA reagent | OpuAC-V360C/ N423C | This paper | Expression plasmid for OpuAC(V360C/N423C) from the pNZOpuCHis construct | |
Recombinant DNA reagent | OppA-A209C/ S441C | This paper | Expression plasmid for OppA(A209C/ S441C) from the pNZcLIC-OppA construct | |
Sequence- based reagent | Primers | Merck | see Supplementary File 2 | |
Peptide, recombinant protein | RPPGFSPFR | Merck | Cat#:B3259 | peptide sequence: RPPGFSPFR |
Peptide, recombinant protein | RDMPIQAF | CASLO ApS | peptide sequence: RDMPIQAF | |
Peptide, recombinant protein | SLSQSKVLPVPQ | CASLO ApS | peptide sequence: SLSQSKVLPVPQ | |
Peptide, recombinant protein | SLSQSKVLP | CASLO ApS | peptide sequence: SLSQSKVLP | |
Chemical compound, drug | Glycine Betaine | Merck | Cat#:B3501 | |
Chemical compound, drug | Carnitine | Merck | Cat#:94954 | |
Chemical compound, drug | Maltose | Merck | Cat#:63418 | |
Chemical compound, drug | Maltotriose | Merck | Cat#:851493 | |
Chemical compound, drug | Maltotetraose | Carbosynth Limited | Cat#:OM06979 | |
Chemical compound, drug | Maltopentaose | Merck | Cat#:M8128 | |
Chemical compound, drug | Maltohexaose | Santa Cruz Biotechnology | Cat#:sc-218665 | |
Chemical compound, drug | Maltoheptaose | Carbosynth Limited | Cat#:OM06868 | |
Chemical compound, drug | Maltodecaose | Carbosynth Limited | Cat#:OM146832 | |
Chemical compound, drug | Maltooctaose | Carbosynth Limited | Cat#:OM06941 | |
Chemical compound, drug | Beta Cyclodextrin | Merck | Cat#:C4767 | |
Chemical compound, drug | Maltotetroitol | Carbosynth Limited | Cat#:OM02796 | |
Chemical compound, drug | Maltotriitol | Merck | Cat#:M4295 | |
Chemical compound, drug | 3H-Asparagine | American Radiolabeled Chemicals | Cat#:ART 0500–250 µCi | |
Chemical compound, drug | 14C-Glutamine | PerkinEllmer | Cat#:NEC451050UC | |
Chemical compound, drug | 14C-Histidine | PerkinEllmer | Cat#:NEC277E050UC | |
Chemical compound, drug | 14C-Arginine | Moravek | Cat#:MC 137 | |
Chemical compound, drug | 3H-Lysine | PerkinEllmer | Cat#:NET376250UC | |
Chemical compound, drug | Alexa555 | Thermo Fisher Scientific | Cat#:A20346 | |
Chemical compound, drug | Alexa647 | Thermo Fisher Scientific | Cat#:A20347 | |
Chemical compound, drug | Cy3B | GE Healthcare | Cat#:PA63131 | |
Chemical compound, drug | ATTO647N | ATTO-TECH | Cat#:AD 647 N-45 | |
Software, algorithm | Dual-Channel- Burst-Search | DOI: 10.1021/ jp063483n | ||
Software, algorithm | LabView data acquisition | DOI: 10.1371/journal. pone.0175766 | Provided by Shimon Weiss, UCLA | |
Software, algorithm | Hidden Markov Model | DOI: 10.1109/ 5.18626 | ||
Software, algorithm | Origin | OriginLab | RRID:SCR_002815 | |
Software, algorithm | MATLAB | MathWorks | RRID:SCR_001622 |
P-values of two-way Kolmogorov-Smirnov test on the solution-based smFRET data.
Primer sequences of all protein constructs used in this study.
Apparent FRET efficiency values of solution-based measurements.
Statistics of confocal scanning experiments of immobilized molecules.