Microsecond sub-domain motions and the folding and misfolding of the mouse prion protein
- Tata Institute of Fundamental Research, India
- Indian Institute of Science Education and Research, India
Figures
Figure 1 with 1 supplement
Structure of moPrP.
Full length moPrP (23-231) is shown (PDB ID: 1AG2). The two distances, W144/C199 and W171/C225, across which the structural dynamics were monitored by PET-FCS are indicated by yellow arrows. For …
Figure 1—figure supplement 1
Far-UV CD spectra of different labeled and unlabeled mutant variants of moPrP.
The CD spectra of (a) W144/C199 moPrP, (b) Trp-less control protein C199 moPrP, (c) W171/C225 moPrP, and (d) Trp-less control protein C225 moPrP are shown. The spectra of the unlabeled and labeled …
Figure 2 with 3 supplements
Microsecond dynamics of moPrP at pH 4 monitored by PET-FCS.
(a) The autocorrelation function (ACF) of W144/C199-Atto moPrP is shown in red. The ACF of free Atto dye, acquired under identical conditions, is shown in blue. The ACF of the control Trp-less …
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Figure 2—source data 1
ACFs and fluorescence spectra of moPrP mutant variants, control Trp-less proteins and dye.
- https://doi.org/10.7554/eLife.44766.011
Figure 2—figure supplement 1
Residuals of fits to the ACF to Equation 1 in main text.
(a) Residuals for the fit to the ACF of W144/C199-Atto moPrP (The data and fit are shown in Figure 2a). (b) Residuals of the fit to the ACF of W171/C225-Atto moPrP (The data and fit are shown in Figu…
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Figure 2—figure supplement 1—source data 1
Residuals of fits to the ACF.
- https://doi.org/10.7554/eLife.44766.006
Figure 2—figure supplement 2
ACF of W144/C199-Atto moPrP in 6 M urea at pH 4.
The raw data are shown in red and fit to the data to the equation having a diffusion component and a single exponential component is shown in black. The diffusion time obtained is 955 µs and the …
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Figure 2—figure supplement 2—source data 1
ACF of W144/C199-Atto moPrP in6Murea at pH 4.
- https://doi.org/10.7554/eLife.44766.008
Figure 2—figure supplement 3
Equilibrium unfolding transition of Atto655-labeled mutant variants of moPrP.
(a) Equilibrium unfolding transition of W144/C199-Atto moPrP, monitored by measuring the fluorescence of Atto 655 moiety attached to the protein is shown. The continuous line through the data points …
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Figure 2—figure supplement 3—source data 1
Equilibrium unfolding transition of Atto655-labeled mutant variants of moPrP.
- https://doi.org/10.7554/eLife.44766.010
Figure 3 with 1 supplement
Folding/unfolding kinetics of W144/C199-Atto moPrP monitored by microsecond mixing experiments.
(a) Unfolding kinetic traces at 3.25 M urea and 4.9 M urea are shown in red and blue, respectively. (b) Refolding kinetic traces at 1.9 M urea (red) and 2.3 M urea (blue) are shown. (c) A comparison …
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Figure 3—source data 1
Folding/unfolding kinetics of W144/C199-Atto moPrP monitored by microsecond mixing experiments.
- https://doi.org/10.7554/eLife.44766.017
Figure 3—figure supplement 1
Refolding kinetic trace of W144/C199-Atto moPrP at 1.9 M Urea concentration.
(a) The fit to a single exponential equation is shown in black. The residuals are plotted below the plot. (b) The fit to a double exponential equation is shown in black and the residuals are shown …
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Figure 3—figure supplement 1—source data 1
Refolding kinetic trace of W144/C199-Atto moPrP at1.9MUrea concentration along with the residuals to fit to a single and double exponetial equation.
- https://doi.org/10.7554/eLife.44766.016
Figure 4
Folding scheme of moPrP at pH 4.
The folding scheme is based on the data obtained from PET-FCS and microsecond mixing experiments. The faster exponential components in the ACFs of PET-FCS experiments have been attributed to native …
Figure 5 with 1 supplement
Effect of salt on the microsecond dynamics of moPrP at pH 4.
(a) ACFs of W144/C199-Atto moPrP are shown, in the absence (red line) and in the presence (blue line) of 150 mM NaCl. (b) ACFs of W171/C225-Atto moPrP are shown, in the absence (red line) and the …
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Figure 5—source data 1
Effect of salt on the microsecond dynamics of moPrP at pH 4.
- https://doi.org/10.7554/eLife.44766.022
Figure 5—figure supplement 1
Effect of salt on the stability of moPrP.
Equilibrium unfolding transitions of W144/C200-Atto moPrP in the presence of 150 mM NaCl (blue data points), and in the absence of salt (red data points). Equilibrium unfolding experiments were …
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Figure 5—figure supplement 1—source data 1
Equilibrium unfolding transitions of W144/C200-Atto moPrP in the presence and absence of salt.
- https://doi.org/10.7554/eLife.44766.021
Figure 6
Rate constants of contact (complex) formation and dissociation within the native state ensemble of moPrP at pH 4.
The rate constants were calculated for the N to N** transition using equilibrium constant K2 and time constant τ2 obtained from PET FCS ACFs. The rate constants of (a) contact formation and (b) …
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Figure 6—source data 1
Rate constants of contact (complex) formation and dissociation within the native state ensemble of moPrP at pH 4.
- https://doi.org/10.7554/eLife.44766.025
Figure 7
Schematic free-energy diagram of moPrP showing the conformational fluctuations in the absence (continuous line) and in the presence of salt (dashed line).
The free energy diagram is constructed based on the values of thermodynamic parameters listed in Table 3. The U ↔ I transitions are described by the time constants of the slowest exponential process …
Tables
Table 1
Parameters obtained from the ACFs of W144/C199-Atto moPrP and W171/C225-Atto moPrP.
The parameters defining the dynamics in the absence of added salt were obtained from the fits to the data shown in Figure 2a and b. The errors shown are the standard deviations determined from …
| W144/C199-Atto moPrP | W171/C225-Atto moPrP | |||
|---|---|---|---|---|
| pH 4 | pH 4, 150 mM NaCl | pH 4 | pH 4, 150 mM NaCl | |
| K1 | 1.2 ± 0.1 | 0.9 ± 0.2 | 0.3 ± 0.07 | 0.65 ± 0.08 |
| K2 | 0.8 ± 0.4 | 1.3 ± 0.2 | 0.4 ± 0.1 | 0.3 ± 0.1 |
| K3 | 0.4 ± 0.02 | 0.2 ± 0.02 | 0.65 ± 0.3 | 0.25 ± 0.1 |
| τ1 (µs) | 1.1 ± 0.3 | 0.8 ± 0.1 | 1 ± 0.8 | 0.5 ± 0.06 |
| τ2 (µs) | 5 ± 2 | 3 ± 0.2 | 19 ± 3 | 4 ± 1 |
| τ3 (µs) | 80 ± 11 | 61 ± 28 | 122 ± 11 | 89 ± 24 |
| τD (µs) | 306 ± 26 | 321 ± 20 | 277 ± 3 | 323 ± 20 |
Table 2
Comparison of equilibrium constants between the dark (N*+N**) states and the fluorescent (N) state obtained from fluorescence spectra and PET-FCS.
The ratio of the fluorescence intensity of the Trp-containing PET construct to that of the corresponding Trp-less control protein (Figure 2c and d), which is equal to 1 + K1+K2, was used to …
| (K1 + K2) calculated from fluorescence spectra | (K1 + K2) obtained from ACFs of PET-FCS | |
|---|---|---|
| W144/C199-Atto moPrP | 1.8 ± 0.2 | 2 ± 0.5 |
| W171/C225-Atto moPrP | 0.5 ± 0.2 | 0.7 ± 0.2 |
Table 3
Effect of salt on thermodynamic parameters governing the dynamics of W144/C199-Atto moPrP.
The equilibrium constants were obtained from the amplitudes of ACFs obtained from PET-FCS experiments. Note that KNN*=N*/N, KNN**=N**/N and KUI = I/U. ΔGUI is calculated from the value of KUI. The …
| W144/C199-Atto moPrP | ||
|---|---|---|
| pH 4 | pH 4, 150 mM salt | |
| KNN* (K1) | 1.2 ± 0.1 | 0.9 ± 0.2 |
| KNN** (K2) | 0.8 ± 0.4 | 1.3 ± 0.2 |
| KUI (K3) | 0.4 ± 0.02 | 0.2 ± 0.02 |
| ΔG UI (kcal/mol) | 0.5 ± 0.1 | 0.9 ± 0.1 |
| ΔG (N+N*+N**)I (kcal/mol) | 4.4 ± 0.2 | 4.1 ± 0.2 |
| ΔG(N+N*+N**)U (kcal/mol) | 3.9 ± 0.3 | 3.2 ± 0.3 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (E. coli BL21 Star (DE3)) | E. coli BL21 DE3* | Thermo Fisher scientific | ||
| Recombinant DNA reagent (plasmid pET 22b) | Plasmid expressing W144/C199 moPrP | Generated by Dr. Ishita Sengupta (Udgaonkar lab) | The results of the publication are under communication | |
| Recombinant DNA reagent (plasmid pET 22b) | Plasmid expressing Trp-less control protein C199 moPrP | This paper | This construct was made by site directed mutagenesis | |
| Recombinant DNA reagent (plasmid pET 22b) | Plasmid expressing W171/C225 moPrP | This paper | This construct was made by site directed mutagenesis | |
| Recombinant DNA reagent (plasmid pET 22b) | Plasmid expressing Trp-less control protein C225 moPrP | This paper | This construct was made by site directed mutagenesis | |
| Software, algorithm | SigmaPlot | Systat Software Inc | ||
| Software, algorithm | Symphotime-64 | PicoQuant |
Additional files
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Supplementary file 1
Dependence of the parameters obtained from the PET-FCS ACFs on excitation power.
The parameters listed were obtained by fitting the ACFs to Equation 1 (Materials and Methods). The experiment was carried out using the W144/C199-Atto moPrP variant at pH 7, in the presence of 150 mM salt. The excitation power was measured from the counts from a calibrated photodiode placed before the main dichroic mirror.
- https://doi.org/10.7554/eLife.44766.027
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Supplementary file 2
Parameters obtained from the ACFs of W144/C199-Atto moPrP and W171/C225-Atto moPrP at pH 7.
- https://doi.org/10.7554/eLife.44766.028
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Transparent reporting form
- https://doi.org/10.7554/eLife.44766.029
