Tracking multiple conformations occurring on angstrom-and-millisecond scales in single amino-acid-transporter molecules
- Department of Physiology, Perelman School of Medicine, University of Pennsylvania, United States
Figures
Figure 1 with 1 supplement
Illustration of the attachment of fluorophore to the AdiC protein and the protein to a glass surface.
(A, B) The orientation of the fluorophore dipole, defined in in terms of θ and φ, can be directly related to that of the alpha helix (A), to which bifunctional rhodamine is attached via two mutant …
Figure 1—figure supplement 1
Gel filtration chromatography and ITC profiles.
(A, B) Size-exclusion chromatography profiles of AdiC protein without (A; Superderx 200 10/300 column) and with (B; Superose 6 10/300 column) the G188C-S195C double cysteine mutations. The elution …
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Figure 1—figure supplement 1—source data 1
Data for gel filtration chromatography and ITC profiles.
- https://cdn.elifesciences.org/articles/82175/elife-82175-fig1-figsupp1-data1-v2.xlsx
Figure 2
Schematic for four polarized emission intensities collected via a microscope and imaged on an EMCCD camera.
Photons, emitted from a fluorophore excited by a circularly polarized laser beam, are collected by an objective and directed to a non-polarizing beam splitter (NPBS) that splits it evenly to two …
Figure 3 with 2 supplements
Polarized intensity components of single fluorescent particles and θ and φ angles calculated from the components.
(A) Consecutive frames of four intensity components (I0, I45, I90 and I135) of a bifunctional-rhodamine-labeled apo AdiC molecule captured over a 300 ms interval in a 7.2 s recording (Figure 3—video …
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Figure 3—source data 1
Intensity and angle data for [Arg]=0 mM condition.
- https://cdn.elifesciences.org/articles/82175/elife-82175-fig3-data1-v2.xlsx
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Figure 3—source data 2
Intensity and angle data for [Arg]=0.75 mM condition.
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Figure 3—figure supplement 1
Total intensities of individual molecules including the bleaching step.
The shown intensities are the same as those Itot traces shown in Figure 3B and C except being 10% longer. The small extra length of each trace contains the bleaching step and is not used in the data …
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Figure 3—figure supplement 1—source data 1
Intensity data including the bleaching step for [Arg]=0 mM condition.
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Figure 3—figure supplement 1—source data 2
Intensity data including the bleaching step for [Arg]=0.75 mM condition.
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Figure 3—video 1
A 7.2 second-long video of the four polarized emission intensities captured on an EMCCD camera, ordered top to bottom as I0, I45, I90 and I135.
A portion of these intensities are also shown in Figure 3A. Running traces of the intensities integrated from the original movies are displayed in the middle section, and the angle traces, θ, φ and Ω…
Figure 4
Experimental resolution of conformational states.
(A, B) The σ values of the θ (A) and φ (B) populations, built with data acquired from individual single AdiC molecules labeled with bifunctional rhodamine, are plotted against SNR.
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Figure 4—source data 1
Data for the relation between SNR and σ of φ and θ.
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Figure 5
Ensemble 3D probability density distributions of θ and φ.
The θ and φ distributions of four individual states in the absence or presence of the indicated concentrations of Arg+, in which the value of φ is plotted along the x-axis, the value of θ along the …
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Figure 5—source data 1
φ angles for probability density distributions, organized by state and [Arg+].
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Figure 5—source data 2
θ angles for probability density distributions, organized by state and [Arg+].
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Figure 5—source data 3
φ and θ sample histogram data for [Arg+] = 0 mM.
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Figure 5—source data 4
φ and θ sample histogram data for [Arg+] = 0.75 mM.
- https://cdn.elifesciences.org/articles/82175/elife-82175-fig5-data4-v2.xlsx
Figure 6
Angle values of individual conformational states.
The values of θ and φ (mean ± sem) for each of the four conformations are plotted against the concentration of Arg+. The number of events is 691–3084. The symbols for the conformational state C1 is …
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Figure 6—source data 1
Table of mean values for θ and φ organized by state and [Arg+].
- https://cdn.elifesciences.org/articles/82175/elife-82175-fig6-data1-v2.xlsx
Figure 7 with 1 supplement
The orientations of the dipole vector of the fluorescence probe in different conformational states.
(A–D) The positions of the arrowheads of individual dipole vectors are mapped onto a unit sphere, on the basis of being in a Cartesian coordinate system defined according to the usual laboratory …
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Figure 7—source data 1
Orientation information of the fluorophore for [Arg] = 0 mM.
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Figure 7—source data 2
Orientation information of the fluorophore for [Arg] = 0.75 mM.
- https://cdn.elifesciences.org/articles/82175/elife-82175-fig7-data2-v2.xlsx
Figure 7—figure supplement 1
Transformation between the laboratory and local coordinates, and assignment of states.
(A) Coordinate transformation. In the laboratory frame of reference (colored black), the zlab-axis is defined as parallel to the optical axis of the microscope objective and the xlab,ylab-axes are …
Figure 8
Comparison of the orientations of helix 6A in the corresponding states determined from the crystal structures and in the polarization study.
(A) Depiction of the six Ω angles among the four orientations of the helix in the four states, represented by four arrows color-coded for states. (B, C) The θ and φ angles (B) of the helix for …
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Figure 8—source data 1
θ and φ values of the four conformational states.
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Figure 8—source data 2
Ω values of the four conformational states.
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Figure 9
3D probability density distributions of mean θ and φ determined from individual molecules.
The value of φ is plotted along the x-axis, the value of θ along the y-axis, and the value of probability density along the z-axis, obtained in the absence (top) and presence (bottom) of a …
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Figure 9—source data 1
φ data for mean angle distributions.
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Figure 9—source data 2
θ data for mean angle distributions.
- https://cdn.elifesciences.org/articles/82175/elife-82175-fig9-data2-v2.xlsx
Figure 10
Ligand dependence of the probabilities of conformational states and the diagram of a conformational state model of AdiC.
(A) The probabilities of individual states (mean ± sem, number of events is 691–3084) are plotted against the Arg+ concentration on a logarithm scale. The four curves superimposed on the data …
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Figure 10—source data 1
State probabilities and associated errors organized according to [Arg+].
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Figure 11
Relations between the structural and conformational states determined respectively from the crystal structures and in the polarization study.
(A, B) Alignments of AdiC’s helix 6A in the structural states EO (blue) (PDB: 7O82) and EX (yellow) (PDB: 3L1L) with the corresponding helices of BasC and ApcT in the states IO (orange) (PDB: 6F2G) …
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Figure 11—source data 1
Tables comparing mean angles calculated from polarization measurements and those from structure.
Table listing the inverse values of combined least-distance-squares of the different state combinations.
- https://cdn.elifesciences.org/articles/82175/elife-82175-fig11-data1-v2.xlsx
Tables
Table 1
Probabilities and equilibrium constants for apo and Arg+ bound states.
| Apo state | ||||||
|---|---|---|---|---|---|---|
| P1 | 0.192 + 0.015/–0.024 | |||||
| K2,1 | 2.566 + 0.589/–0.244 | P2 | 0.492 + 0.040/–0.014 | |||
| K3,1 | 0.923 + 0.061/–0.149 | P3 | 0.177 + 0.011/–0.037 | |||
| K4,1 | 0.729 + 0.225/–0.103 | P4 | 0.140 + 0.026/–0.014 | |||
| Arg+ bound state | KD (µM) | |||||
| argP1 | 0.172 + 0.020/–0.020 | argKD1 | 49 + 46/–25 | |||
| argK2,1 | 1.795 + 0.298/–0.319 | argP2 | 0.308 + 0.013/–0.025 | argKD2 | 69 + 67/–33 | |
| argK3,1 | 1.782 + 0.272/–0.193 | argP3 | 0.306 + 0.019/–0.018 | argKD3 | 25 + 20/–14 | |
| argK4,1 | 1.250 + 0.228/–0.188 | argP4 | 0.214 + 0.018/–0.015 | argKD4 | 28 + 31/–14 | |
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Parameters from fit of Equation 5 to the plots of AdiC conformational state probabilities versus [Arg+] (Figure 10A). 95% confidence intervals were calculated from the maximum likelihood optimization of 1000 sets of simulated data obtained from bootstrapping.
