1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Conformational and dynamic plasticity in substrate-binding proteins underlies selective transport in ABC importers

  1. Marijn de Boer
  2. Giorgos Gouridis
  3. Ruslan Vietrov
  4. Stephanie L Begg
  5. Gea K Schuurman-Wolters
  6. Florence Husada
  7. Nikolaos Eleftheriadis
  8. Bert Poolman  Is a corresponding author
  9. Christopher A McDevitt  Is a corresponding author
  10. Thorben Cordes  Is a corresponding author
  1. University of Groningen, The Netherlands
  2. Ludwig-Maximilians-Universität München, Germany
  3. KU Leuven, Belgium
  4. University of Melbourne, Australia
  5. The University of Adelaide, Australia
Research Article
Cite this article as: eLife 2019;8:e44652 doi: 10.7554/eLife.44652
7 figures, 3 tables and 5 additional files

Figures

Representative SBPs from different structural clusters, categorized by their hinge region.

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 open, ligand-free conformation. Hinge regions are shown in blue and the two rigid lobes in grey. For classification of the proteins in clusters see (Berntsson et al., 2010; Scheepers et al., 2016).

https://doi.org/10.7554/eLife.44652.002
Figure 2 with 4 supplements
Conformational states of SBPs probed by smFRET reveal multiple active conformations.

(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), SBD1(T159C/G87C) (E), SBD2(T369C/S451) (F) and OppA(A209C/S441C) (G) in the absence (grey bars) and presence of different cognate substrates (green bars). The OppA substrates are indicated by one-letter amino acid code. Bars are the data and the solid line a Gaussian fit. The 95% confidence interval of the Gaussian distribution mean is shown in Supplementary file 3, and the interval center is indicated by vertical lines (solid and dashed). (H) Mean of the Gaussian distribution of MalE labeled at T36/S352 (black), T36/N205 (green) or K34/R352 (blue). Error bars indicate 95% confidence interval.

https://doi.org/10.7554/eLife.44652.005
Figure 2—source data 1

Apparent FRET efficiency histograms of Figure 2B–G.

https://doi.org/10.7554/eLife.44652.010
Figure 2—source data 2

Apparent FRET efficiency histograms of Figure 2—figure supplement 3.

https://doi.org/10.7554/eLife.44652.011
Figure 2—figure supplement 1
Ligand-induced conformational dynamics of SBPs.

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), SBD1(T159C/G87C) (D) and OppA(A209C/S441C) (E) in the presence of the indicated substrate concentration. In the fluorescence trajectories: the top panel shows the calculated apparent FRET efficiency (blue) from the donor (green) and acceptor (red) photon counts as shown in the bottom panels. The most probable state-trajectory of the Hidden Markov Model (HMM) is shown by the orange line. Statistics in Supplementary file 4. The histogram was fitted with two Gaussian distribution to obtain the relative population of the high FRET state P. Ignoring the small contribution of intrinsic closing (Figure 3H), we use KD=L (1-P)/P (one site-binding model), where L is the indicated ligand concentration, to determine KD (see Table 1).

https://doi.org/10.7554/eLife.44652.006
Figure 2—figure supplement 2
OppA uses an induced-fit ligand binding mechanism.

(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 apparent FRET efficiency (blue) with the most probable state-trajectory of the Hidden Markov Model (HMM) (orange). Dwell time histogram of the high FRET (closed conformation) (B) and low FRET state (open conformation) (C) as obtained from the most probable state-trajectory of the HMM. Bars are the data and the solid line is an exponential fit. Statistics in Supplementary file 4. (D) Average closing rate (rate of low to high FRET state; black) and average lifetime of the ligand-bound conformation (lifetime high FRET state; purple). Data correspond to mean ± s.e.m. and the solid line a linear fit. Slope or intercept of the fit are shown (95% confidence interval). From the fit a KD of 14 ± 5 µM (95% confidence interval) is obtained. (E) Isothermal calorimetry binding isotherm of the titration of OppA with RPPGFSFR, obtaining KD of 5 ± 3 µM (mean ± s.d., n = 3). Points are the data and the solid line a fit to a one site-binding model.

https://doi.org/10.7554/eLife.44652.007
Figure 2—figure supplement 3
Translocation competent conformation(s) of MalE and OppA.

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 indicated by one-letter amino acid code. Bars are the data and solid line a Gaussian fit. The 95% confidence interval for the mean of the Gaussian distribution is shown in Supplementary file 3, and the interval center is indicated by vertical lines (solid and dashed).

https://doi.org/10.7554/eLife.44652.008
Figure 2—figure supplement 4
MalE conformations studied by smFRET.

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 data and the solid line a Gaussian fit. The 95% confidence interval for the mean of the Gaussian distribution is shown in Supplementary file 3, and the interval center is indicated by vertical lines (solid and dashed). Structure of ligand-free MalE (PDB ID: 1OMP) with corresponding donor and acceptor fluorophore positions is indicated above the histograms.

https://doi.org/10.7554/eLife.44652.009
Figure 3 with 2 supplements
Rare conformational states of ligand-free SBPs.

(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), MalE(T36C/S352C) (D), SBD1(T159C/G87C) (E), OppA(A209C/S441C) (F) and SBD2(T369C/S451) (G) in the absence of substrate. 10–20 μM of unlabeled protein or 1 mM EDTA (for PsaA) was added to scavenge any ligand contaminations. In all fluorescence trajectories presented in the figure: top panel shows calculated apparent FRET efficiency (blue) from the donor (green) and acceptor (red) photon counts as shown in the bottom panels. Orange lines indicate average apparent FRET efficiency value or most probable state-trajectory of the Hidden Markov Model (HMM). Statistics in Supplementary file 4. (H) Percentage of time a SBP is in the high FRET state. Statistics in Supplementary file 4.

https://doi.org/10.7554/eLife.44652.012
Figure 3—source data 1

Donor and acceptor photon counts, apparent FRET efficiency and most probable state-trajectory of the Hidden Markov Model of the traces in Figure 3.

https://doi.org/10.7554/eLife.44652.015
Figure 3—figure supplement 1
Conformational dynamics of ligand-free and ligand-bound SBPs.

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 substrate and under saturating conditions of ligand, as indicated. In the absence of ligand, 10–20 μM of unlabeled protein or 1 mM EDTA (for PsaA) was added to scavenge any ligand contaminations. The top panels show the calculated apparent FRET efficiency (blue) from the donor (green) and acceptor (red) photon counts as presented in bottom panels. The orange line indicates the average apparent FRET efficiency value or most probable state-trajectory of the HMM. Statistics in Supplementary file 4.

https://doi.org/10.7554/eLife.44652.013
Figure 3—figure supplement 2
Intrinsic conformational dynamics in the presence of unlabeled protein.

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 potential ligand contaminations. Examples of the high FRET transitions are shown in Figure 3 and Figure 3—figure supplement 1. Error bars correspond to s.e.m. The closing rate was determined by dividing the total observation time of all molecules by the number of observed high FRET transitions. The statistical significance of the average closed state lifetime was determined by a two-tailed unpaired t-tests. The statistical significance of the closing rate was determined by testing for the difference in the proportion of time-bins in which a low to high FRET transition is made and using the z-test. Statistics in Supplementary file 4.

https://doi.org/10.7554/eLife.44652.014
Figure 4 with 3 supplements
Substrate-specificity of GlnPQ and SBP conformations induced by non-cognate substrates.

(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 trans with a plasmid for expressing GlnPQ; the final amino acid concentrations are indicated between brackets. Points are the data and the solid line a hyperbolic fit. Time-dependent uptake of glutamine (B) and asparagine (C) in proteoliposomes reconstituted with purified GlnPQ (see Materials and methods section). The final concentration of [14C]-glutamine and [14C]-asparagine was 5 μM, respectively; the amino acids indicated in the panel were added at a concentration of 5 mM. Solution-based apparent FRET efficiency histogram of SBD1(T159C/G87C) (D), SBD2(T369C/S451) (E), MalE(T36C/S352C) (F), OpuAC(V360C/N423C) (G) and PsaA(V76C/K237C) (H) in the presence of non-cognate (red bars) substrates as indicated. Bars are the data and solid line a Gaussian fit. The 95% confidence interval for the distribution mean is shown in Supplementary file 3. The interval center is indicated by vertical lines (solid and dashed).

https://doi.org/10.7554/eLife.44652.016
Figure 4—source data 1

Apparent FRET efficiency histograms of Figure 4D–H.

https://doi.org/10.7554/eLife.44652.020
Figure 4—figure supplement 1
Substrate binding of SBD1 and SBD2 studied by ensemble FRET.

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 7.4. Amino acids are indicated by their three letter abbreviation. Data correspond to mean ± s.d. of the apparent FRET change of duplicate measurements with the same labeled protein sample.

https://doi.org/10.7554/eLife.44652.017
Figure 4—figure supplement 2
Non-cognate substrate binding by SBD1 and SBD2.

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 solid lines a fit to a mixture model with two Gaussian distributions or a fit with a single Gaussian distribution. The mean of the Gaussian distributions was obtained from the extreme conditions and fixed in the mixture model. Fraction of SBD1 bound to asparagine (D), SBD2 bound to glutamine (E) and SBD1 bound to histidine (F). Points are the data and the solid line a fit to a one site-binding model. (G) Estimated dissociation constants KD as obtained from the fit. Error bars represent 95% confidence interval.

https://doi.org/10.7554/eLife.44652.018
Figure 4—figure supplement 3
PsaA(E74C/K237C) conformational changes probed by smFRET.

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 for the distribution mean is shown in Supplementary file 3. The interval center is indicated by vertical lines (solid and dashed).

https://doi.org/10.7554/eLife.44652.019
Opening transition in PsaA dictates transport specificity.

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 for the indicated duration. Bars are the data and the solid line a Gaussian fit. The 95% confidence interval for the mean of the Gaussian distribution can be found in Supplementary file 3, and the interval center is indicated by vertical lines (solid, metal-free and dashed, metal-bound). (D) Whole cell Zn2+ accumulation of S. pneumoniae D39 and mutant strains in CDM supplemented with 50 µM ZnSO4 as determined by ICP-MS. Data correspond to mean ± s.d. μg Zn2+.g−1 dry cell weight from three independent biological experiments. Statistical significance was determined by one-way ANOVA with Tukey post-test (***p < 0.005 and ****p < 0.0001).

https://doi.org/10.7554/eLife.44652.021
Figure 6 with 3 supplements
Lifetime of MalE ligand-bound conformations and relation to activity.

(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 mean ± s.e.m. Data in Figure 6—figure supplement 2. Statistical significance was determined by two-tailed unpaired t-tests (***p < 0.005 and ****p < 0.0001). (B, C, D, E, F and G) Representative fluorescence trajectories of MalE(T36C/S352C) in the presence of different substrates as indicated. In all fluorescence trajectories presented: top panel shows calculated apparent FRET efficiency (blue) from the donor (green) and acceptor (red) photon counts as shown in the bottom panels. Most probable state-trajectory of the Hidden Markov Model (HMM) is shown (orange). (H) Published ATPase activity (Hall et al., 1997a) linked to the lifetime of the closed MalE conformation induced by transport of different cognate substrates as indicated. Points are the data and the solid line a simple linear regression fit.

https://doi.org/10.7554/eLife.44652.022
Figure 6—source data 1

Lifetimes of the high FRET state of the data shown in Figure 6A and Figure 6—figure supplement 2.

https://doi.org/10.7554/eLife.44652.026
Figure 6—source data 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.

https://doi.org/10.7554/eLife.44652.027
Figure 6—source data 3

Lifetimes of the high FRET state of the data shown in Figure 6—figure supplement 3B.

https://doi.org/10.7554/eLife.44652.028
Figure 6—figure supplement 1
Surface-based smFRET histogram of MalE.

(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 Model (HMM), the apparent FRET efficiencies of the low (ligand-free conformation) and high FRET state (closed ligand-bound conformation) were obtained. The final histogram was constructed from all fluorescence trajectories. Representative fluorescence trajectories are shown in Figure 6B–G. Bars are the data and solid line a Gaussian fit. The 95% confidence interval for the distribution mean is indicated. The average apparent FRET efficiency of the solution-based smFRET measurements (Figure 2—figure supplement 3A) is indicated by vertical lines.

https://doi.org/10.7554/eLife.44652.023
Figure 6—figure supplement 2
Lifetime distribution of the ligand-bound conformations of MalE.

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 in Figure 6B–G. Grey bars are the data and the solid line an exponential fit. Statistics in Supplementary file 4.

https://doi.org/10.7554/eLife.44652.024
Figure 6—figure supplement 3
Conformational changes and dynamics of MalE(A96W/I329W).

(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 (blue) from the donor (green) and acceptor (red) photon counts as shown in the bottom panel. The most probable state-trajectory of the Hidden Markov Model (HMM) is shown (orange). (B) Dwell time histogram of the high FRET state (closed conformation) as obtained from the most probable state-trajectory of the HMM of all molecules. Grey bars are the data and the solid line is an exponential fit. Statistics in Supplementary file 4. (C) Solution-based apparent FRET efficiency histogram of MalE and MalE(A96W/I329W) in the presence of 1 mM maltose for the indicated inter-dye positions. Bars are the data and solid line a Gaussian fit. The 95% confidence interval for the mean of the Gaussian distribution is indicated. The FRET distributions of the wildtype and mutant protein are not significantly different; p = 0.28 (T36C/S352C) and p = 0.30 (K34C/R352) using the two-way KS test.

https://doi.org/10.7554/eLife.44652.025
The conformational changes and dynamics of SBPs and the regulation of transport.

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 are bound via induced fit (‘ligand-binding via induced fit’). SBPs can acquire one or more conformations that can activate transport (‘multiple conformations activate transport’). Variations in cognate substrate transport are caused by: (i) openings rate of the SBP and substrate transfer to the translocator (‘faster SBP opening – faster transport’) and (ii) substrate-dependent downstream steps (‘kinetics of downstream steps are substrate-dependent’). Although SBPs can acquire a conformation that activates transport (‘conformational match’), transport still fails when: (i) the SBP has no affinity for the translocator and/or cannot make the allosteric interaction with the translocator (‘conformational mismatch’); (ii) the SBP cannot open and release the substrate to the translocator (‘SBP cannot open’); or (iii) due to the specificity and size limitations of the translocator (‘rejected by translocator’).

https://doi.org/10.7554/eLife.44652.029

Tables

Table 1
Dissociation constant KD of substrate-binding proteins.
https://doi.org/10.7554/eLife.44652.003
KD (µM)
Protein*LigandFreely-diffusing proteinSurface-tethered proteinKD WT protein (µM)
OpuAC(V360C/N423C)Glycine betaine3.4 ± 0.43.14–5 (Wolters et al., 2010)
OppA(A209C/S441C)RPPGFSFR7.0 ± 114 ± 5#5 ± 3#
SBD2(T369C/S451)Glutamine1.2 ± 0.2§0.50.9 ± 0.1 (Gouridis et al., 2015)
SBD1(T159C/G87C)Asparagine0.34 ± 0.03§0.30.2 ± 0.0 (Gouridis et al., 2015)
MalE(T36C/S352C)Maltose1.7 ± 0.32.21-2 (Hall et al., 1997a, Kim et al., 2013)
MalE(T36C/S352C)Maltotriose0.6 ± 0.20.90.2-2 (Hall et al., 1997a, Kim et al., 2013)
  1. *. KD could not be determined reliably for labeled PsaA due to background metal contamination.

    †. Population of the closed conformation P in the presence of a ligand concentration L was determined using solution-based smFRET. The KD=L (1-P)/P for a one-binding site model. Data corresponds to mean ± s.d. of duplicate experiments with the same protein sample.

  2. #. Figure 2—figure supplement 2

    ¶. The KD values of wildtype (WT) proteins are obtained from the indicated references.

Table 2
Steady-state anisotropy values.
https://doi.org/10.7554/eLife.44652.004
Anisotropy
Alexa555Alexa647Cy3BAtto647N
Free dye0.250.200.080.08
OpuAC(V360C/N423C)NANA0.170.11
OppA(A209C/S441C)0.250.19NANA
SBD1(G87C/T159C)0.270.19NANA
SBD2(T369C/S451)0.260.20NANA
MalE(T36C/S352C)0.290.24NANA
PsaA(V76C/K237C)0.280.22NANA
  1. NA: not applicable. Data correspond to mean (s.d. below < 0.01) of duplicate experiments, using the same labeled protein sample.

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene(Escherichia coli)MalENAUniProt:
P0AEX9
AntibodyMouse anti-hisQiagenRRID:AB_2714179(1:200)
Strain,
strain background (Streptococcus pneumoniae)
D39National Collection of Type CulturesNCTC:7466Capsular serotype 2
Strain, strain
background
(Streptococcus
pneumoniae)
D39 ∆psaAThis paperReplacement of psaA with the Janus cassette (∆psaA::Janus)
Strain, strain background (Streptococcus pneumoniae)D39 ∆czcDThis paperReplacement of czcD with the Janus cassette (∆czcD::Janus)
Strain, strain background (Streptococcus pneumoniae)D39 ΩpsaAD280NThis paperReplacement of ∆psaA::Janus with psaA D280N (∆psaA::psaAD280N)
Strain, strain background (Streptococcus pneumoniae)D39 ΩpsaAD280N∆czcDThis paperReplacement of ∆psaA::Janus with psaA D280N; replacement of czcD with the Janus cassette (∆psaA::psaAD280N∆czcD::Janus)
Strain, strain background
(Lactococcus lactis)
NZ9000NIZO food
research
Strain, strain
background
(Lactococcus lactis)
GKW9000DOI: 10.1038/
nsmb2929
Lactococcus lactis NZ9000 with glnPQ gene deleted
Strain, strain
background
(Escherichia coli)
K12OtherProvided by Tassos Economou, KU Leuven
Strain, strain
background
(Escherichia coli)
BL 21 DE3OtherProvided by Tassos Economou, KU Leuven
Recombinant
DNA reagent
pET20bMerckCat#:69739–3
Recombinant
DNA reagent
pNZglnPQhisDOI: 10.1047/
jbc.M500522200
Expression plasmid for GlnPQ
Recombinant
DNA reagent
SBD1-T159C/G87CDOI: 10.1038/
nsmb2929
Expression plasmid for SBD1(T159C/G87C)
Recombinant
DNA reagent
SBD2-T369C/S451CDOI: 10.1038/
nsmb2929
Expression plasmid for SBD2(T369C/S451C)
Recombinant
DNA reagent
pCAMcLIC01-PsaADOI: 10.1038/
nchembio.1382
Expression plasmid for PsaA
Recombinant
DNA reagent
pCAMcLIC01-PsaAD280NDOI: 10.1038/
nchembio.1382
Expression plasmid for PsaA(D280N)
Recombinant
DNA reagent
pNZOpuCHisDOI: 10.1093/
emboj/cdg581
Expression plasmid for OpuAC
Recombinant
DNA reagent
pNZcLIC-OppADOI: 10.1002/pro.97Expression plasmid for OppA
Recombinant
DNA reagent
PsaA-V76C/K237CThis paperExpression plasmid for PsaA(V76C/K237C) from the pCAMcLIC01-PsaA construct
Recombinant
DNA reagent
PsaA-E74C/K237CThis paperExpression plasmid for PsaA(E74C/K237C) from the pCAMcLIC01-PsaA construct
Recombinant
DNA reagent
PsaA-D280N/V76C/K237CThis paperExpression plasmid for PsaA(D280N/V76C/K237C) from the pCAMcLIC01-PsaAD280N construct
Recombinant
DNA reagent
MalE-T36C/S352CThis paperProgenitors: PCR, E. coli gDNA; pET20b vector
Recombinant
DNA reagent
MalE-T36C/N205CThis paperProgenitors: PCR, E. coli gDNA; pET20b vector
Recombinant
DNA reagent
MalE-K34C/
R354C
This paperProgenitors: PCR, E. coli gDNA; pET20b vector
Recombinant
DNA reagent
MalE-T36C/S352C/
A96W/I329W
This paperProgenitors: PCR,
E. coli gDNA;
pET20b vector
Recombinant
DNA reagent
OpuAC-V360C/
N423C
This paperExpression plasmid
for OpuAC(V360C/N423C)
from the pNZOpuCHis
construct
Recombinant
DNA reagent
OppA-A209C/
S441C
This paperExpression plasmid
for OppA(A209C/
S441C) from the
pNZcLIC-OppA
construct
Sequence-
based reagent
PrimersMercksee Supplementary File 2
Peptide,
recombinant
protein
RPPGFSPFRMerckCat#:B3259peptide sequence:
RPPGFSPFR
Peptide,
recombinant
protein
RDMPIQAFCASLO ApSpeptide sequence:
RDMPIQAF
Peptide,
recombinant
protein
SLSQSKVLPVPQCASLO ApSpeptide sequence:
SLSQSKVLPVPQ
Peptide,
recombinant
protein
SLSQSKVLPCASLO ApSpeptide sequence:
SLSQSKVLP
Chemical
compound,
drug
Glycine BetaineMerckCat#:B3501
Chemical
compound,
drug
CarnitineMerckCat#:94954
Chemical
compound,
drug
MaltoseMerckCat#:63418
Chemical
compound,
drug
MaltotrioseMerckCat#:851493
Chemical
compound,
drug
MaltotetraoseCarbosynth
Limited
Cat#:OM06979
Chemical
compound,
drug
MaltopentaoseMerckCat#:M8128
Chemical
compound,
drug
MaltohexaoseSanta Cruz
Biotechnology
Cat#:sc-218665
Chemical
compound,
drug
MaltoheptaoseCarbosynth
Limited
Cat#:OM06868
Chemical
compound,
drug
MaltodecaoseCarbosynth
Limited
Cat#:OM146832
Chemical
compound,
drug
MaltooctaoseCarbosynth
Limited
Cat#:OM06941
Chemical
compound,
drug
Beta
Cyclodextrin
MerckCat#:C4767
Chemical
compound,
drug
MaltotetroitolCarbosynth
Limited
Cat#:OM02796
Chemical
compound,
drug
MaltotriitolMerckCat#:M4295
Chemical
compound,
drug
3H-AsparagineAmerican
Radiolabeled
Chemicals
Cat#:ART 0500–250 µCi
Chemical
compound,
drug
14C-GlutaminePerkinEllmerCat#:NEC451050UC
Chemical
compound,
drug
14C-HistidinePerkinEllmerCat#:NEC277E050UC
Chemical
compound,
drug
14C-ArginineMoravekCat#:MC 137
Chemical
compound,
drug
3H-LysinePerkinEllmerCat#:NET376250UC
Chemical
compound,
drug
Alexa555Thermo Fisher
Scientific
Cat#:A20346
Chemical
compound,
drug
Alexa647Thermo Fisher
Scientific
Cat#:A20347
Chemical
compound,
drug
Cy3BGE HealthcareCat#:PA63131
Chemical
compound,
drug
ATTO647NATTO-TECHCat#: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
OriginOriginLabRRID:SCR_002815
Software,
algorithm
MATLABMathWorksRRID:SCR_001622

Data availability

Data generated or analysed during this study are included in the manuscript and supporting files. Source data files are available for smFRET histogrammes, representative smFRET time-traces and smFRET dwell-time histogrammes as shown in the manuscript. Primer sequences for created protein mutants are included.

Additional files

Supplementary file 1

P-values of two-way Kolmogorov-Smirnov test on the solution-based smFRET data.

https://doi.org/10.7554/eLife.44652.030
Supplementary file 2

Primer sequences of all protein constructs used in this study.

https://doi.org/10.7554/eLife.44652.031
Supplementary file 3

Apparent FRET efficiency values of solution-based measurements.

https://doi.org/10.7554/eLife.44652.032
Supplementary file 4

Statistics of confocal scanning experiments of immobilized molecules.

https://doi.org/10.7554/eLife.44652.033
Transparent reporting form
https://doi.org/10.7554/eLife.44652.034

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