1. Forces Driving Chaperone Action
Philipp Koldewey, Frederick Stull, Scott Horowitz, Raoul Martin, James C.A. Bardwell 
Cell 
Volume 166, Issue 2, Pages (July 2016)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Complex Formation between Spy and Im7A3W Slows Down with Increasing Salt Concentrations as Determined by Stopped-Flow Fluorescence
(A) Representative raw transients for 250 nM Im7A3W mixed with increasing concentrations of Spy at an ionic strength of 0.12 M in the stopped-flow fluorimeter. Traces were fit with a double exponential function to obtain observed rate constants (kobs). The kinetic traces are averages of four replicates.
(B) kobs of the bimolecular step of Spy-Im7 interaction were plotted as a function of Spy dimer concentration to determine the binding (kon) and release (koff) rate constant at increasing ionic strengths and 22°C:  μM Im7A3W (0.045 M), 0.125 μM Im7A3W (0.07 M), 0.250 μM Im7A3W (0.12 M), 0.5 μM Im7A3W (0.22 M), and 1.5 μM Im7A3W (0.32 M) were mixed with increasing concentrations of SpyWT. kobs at low ionic strength (< 0.12 M) were derived from single exponential fits of the raw fluorescence transients, whereas at ionic strength ≥ 0.12 M, double exponential fits were used (see Figure S3). A linear fit of kobs as a function of Spy concentration yielded kon from the slope and koff from the intercept (Table S1). At an ionic strength of 0.12 M, Spy binds to Im7A3W with a kon of 1.2 ± 0.4 × 108 M−1s−1, which is consistent with what was shown for the interaction of Spy with Im7A3 (Stull et al., 2016), demonstrating that the tryptophan substitution does not affect the kinetics of Spy-Im7 interaction. The ionic strength was adjusted with sodium chloride in 40 mM HEPES (pH 7.5). The kobs of four experiment per Spy concentration were plotted to show the experimental error.
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4. Figure 2 Spy-Im7A3W Binding Is Salt-Dependent
(A and B) Stopped-flow binding experiments were conducted in 40 mM HEPES, pH 7.5 of different ionic strengths, adjusted with to 0.30 M sodium chloride (see Figure 1).
(A) The binding rate constant kon of Spy-Im7A3W interaction as a function of ionic strength was derived from the slope of the linear fits of the observed rate constants (see Figure 1B). Errors are propagated fitting standard errors of four independent data points.
(B) The release rate constant koff was derived either from the corrected y-intercepts of linear fits (black, see Figure 1B) or competition experiments (red) (see Figure S3). The binding and release rate constants are affected differently by the ionic strength. Whereas kon decreases exponentially with increasing ionic strength (A), koff increases exponentially (B). Note that at all ionic strengths tested, the release rate constant obtained by binding competition is, within error, identical to the koff determined from the corrected y-intercept. Errors are propagated fitting standard errors of four independent data points.
(C) The binding free energy (ΔG) of Spy-Im7A3W interaction increases exponentially with ionic strength. The ΔG was derived from the kinetic dissociation constant (Kd) (see Table S1). At infinite ionic strength, when all electrostatic interactions are screened, ΔG = −5.2 kcal mol−1, suggesting that hydrophobic interactions contribute to complex stability. Errors are propagated fitting standard errors of three independent data points.
(D) Distribution of positive and negative surface charge on Spy (PDB: 3O39) and folded Im7 (PDB: 1CEI). Whereas positive charges (blue) outweigh negative charges (red) on the concave side of Spy, the convex side reveals a more even charge distribution. In contrast, Im7 contains a hot-spot of condensed negative charge at the site where it binds to its in vivo binding partner E7 (Ko et al., 1999). The electrostatic surface potential was calculated via PyMol using the APBS tools2.1 plugin (a color scale for the charge distribution from −5 to 5 was chosen). The respective .pqr file was generated on the PDB2PQR website for a pH of 7.5 ( (Unni et al., 2011).
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5. Figure 3
Super Spy Variants Q100L and H96L Bind Im7A3W Tighter than SpyWT Due to a Slower Release Rate Constant
(A) Observed rate constants (kobs) of the binding step were derived from single (SpyH96L) double (SpyWT) or triple (SpyQ100L) exponential fits of the raw transients (Figure S3) and are plotted as a function of Spy concentration: 0.25 μM Im7A3W mixed with SpyWT, 1st phase (red), SpyH96L (black), or SpyQ100L, 1st phase (blue). Data were fit to a line to yield the binding rate constant of Spy to Im7 (see Table S1). Three independent data points per Spy concentration were collected to show the experimental error. Note that the kobs of the additional phases can be found in Figure S3.
(B) Binding competition experiments in which 0.25 μM Im7A3W in complex with the respective Spy variant (2 μM SpyWT (red), 2 μM SpyH96L (black), 0.5 μM SpyQ100L (blue)) was mixed with the tryptophan-free, unfolded Im7 variant, Im7A3W75F (see also Figure S3). All traces show a small second phase and had to be fit to a double exponential function. The kinetic traces are averages of four replicates.
All experiments were performed in 40 mM HEPES, pH 7.5, 100 mM sodium chloride. See also Table S1.
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6. Figure 4
Spy-Im7 Interaction Is an Entropy-Driven Process Due to Hydrophobic Interactions in the Complex
(A) Representative ITC binding isotherm of SpyWT + Im7A3W at 22°C. Integrated thermograms (bottom graph) are fit to a single site-binding model.
(B) Binding enthalpy (ΔH) of Spy-Im7 complex formation as a function of temperature measured via ITC. The heat capacity changes (ΔCp) were derived from the slope of a linear fit. Im7A3W binding to SpyWT (red), Spy H96L (black), or Spy Q100L (blue) resulted in a negative ΔCp, whereas Im7WT titrated with SpyWT (magenta) resulted in a positive ΔCp. All experiments were performed in 40 mM HEPES (pH 7.5), 100 mM sodium chloride. Three independent data points per Spy concentration were collected to show the experimental error.
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7. Figure 5
Screening of Ionic Interactions Enthalpically Disfavors Complex Formation
(A and B) ITC binding titrations of Spy-Im7A3W with SpyWT at 22°C were performed in 40 mM HEPES (pH 7.5) containing 25 to 300 mM sodium chloride to obtain thermodynamic parameters: enthalpy (ΔH) (A) and entropy (ΔS) (B). Three independent data points per sodium chloride concentration were collected to show the experimental error.
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8. Figure 6
Native State of Im7 Is Released from Spy 13-Fold Faster than the Unfolded State
Binding competition experiments were performed: 2.5 μM Im7WT in complex with 4 μM SpyWT dimer was mixed with 50 μM of Im7A3W75F to determine the release rate constant of natively folded Im7 (red); 0.25 μM Im7A3W in complex with 0.5 μM SpyWT dimer was mixed with 25 μM of Im7A3W75F to determine the release rate constant of the unfolded state of Im7 (black). In both cases, a double exponential fit was used (see also Figure S7). The second, slow phase observed for Im7WT is caused by either refolding or release of a subpopulation of partially unfolded Im7WT, whereas the fast phase is due to the release of the bound native state of Im7, as revealed by double mixing experiments (see Figure S7). This experiment was performed at 4°C in 40 mM HEPES (pH 7.5) and 25 mM sodium chloride to slow down the release of Im7WT. The kinetic traces are averages of four replicates.
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9. Figure 7 Mechanistic Scheme of Spy-Client Interaction
(1) Client binding rates are maximized through long-range electrostatic attraction, which allows Spy (blue) to effectively compete with aggregation of the unfolded client protein (red). Client release, on the other hand, is energetically disfavored mainly by the solvation of hydrophobic surface area on the client and Spy, which are buried in the complex. (2) Folding of the client results in the burial of hydrophobic residues in the client’s core, which decreases its affinity to Spy, and therefore (3) favors release of the client protein. The electrostatic interactions, however, allow the client to stay bound to Spy while it folds.
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10. Figure S1
Wild-Type Spy Binds Tightly to Unfolded Im7 in a 1:1 Ratio as Determined by Tryptophan Fluorescence and Fluorescence Anisotropy Titrations, Related to Figure 1
Previously, we demonstrated that the endogenous tryptophan W75 of Im7 can be used as a read-out for Im7 folding as well as for Spy binding, as Spy does not contain any tryptophan residues (Stull et al., 2016). It is well-established that the L18AL19AL37A variant of Im7 (Im7A3) mimics the unfolded state of Im7 under non-denaturing conditions (Pashley et al., 2012). However, the endogenous tryptophan W75 of Im7A3 showed only a 12% signal change upon binding to Spy at saturating concentrations (A), making it challenging to characterize the interaction between Im7A3 and Spy kinetically at low protein concentrations using tryptophan fluorescence-based stopped-flow. Therefore, we enhanced the signal upon binding by substituting histidine 40 with a tryptophan to yield Im7 L18AL19AL37AH40W, which we term Im7A3W in this study (B). This substitution increased the signal change upon Spy binding to 150% (B), compared to Im7A3 (A), but did not cause any substantial change in the circular dichroism spectra of the client protein, indicating that Im7A3W, like Im7A3, is completely unfolded in solution (Pashley et al., 2012) (see Figure S1E).
(A and B) Fluorescence titration of Im7A3 or Im7A3W with wild-type Spy to determine the binding affinity (Kd). Binding isotherm of 50 μM Im7A3 (A) or 250 nM Im7A3W (B) titrated with wild-type Spy. The tryptophan was excited at 296 nm and fluorescence emission was recorded at 340 nm. Fitting the fluorescence change as a function of Spy concentration yielded a dissociation constant (Kd) of 10.4 ± 5.8 μM for Im7A3 (A) and 1.4 ± 0.2 μM for Im7A3W (B). All affinities are also listed in Table S1. Similar increases in tryptophan fluorescence of Im7A3 upon addition of Spy were reported previously (Stull et al., 2016). Inset (B): Representative tryptophan fluorescence spectra of Im7A3W titrated with wild-type Spy. The blue shift of the fluorescence maximum and the increase in quantum yield upon addition of increasing quantities of Spy indicate that the tryptophan residues of Im7A3W are less solvent-exposed when it is bound to Spy than when free in solution.
(C and D) The Spy dimer binds to Im7A3 and Im7A3W in a 1:1 complex as determined by fluorescence anisotropy. (C) 50 μM Im7A3 and (D) 10 μM Im7A3W titrated with wild-type Spy. Stoichiometries by fluorescence anisotropy are consistent with other methods presented in this paper: (C) 0.91 ± 0.04; (D) 1.07 ± 0.02; also see Figure S2 and Table S1. Like Im7A3, Im7A3W binds the Spy dimer in a 1:1 ratio, indicating that the tryptophan insertion at position 40 in Im7A3 had no influence on the stoichiometry of the complex. All experiments were performed in 40 mM HEPES (pH 7.5), 100 mM sodium chloride at 22°C. Three independent data points are averaged per Spy concentration to obtain a standard deviation.
(E) Im7A3 (black) and Im7A3W (red) are unstructured as determined by circular dichroism (CD). CD was performed in 40 mM sodium phosphate (pH 7.5) at 22°C.
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11. Figure S2
Spy Binds to Different Unfolded Im7 Variants in a 1:1 Ratio, Related to Figure 1
(A–C) Stoichiometry and molecular mass of wild-type Spy in complex with Im7A3W (A), Im7A3 (B), and Im7A3W75F (C) were determined via sedimentation velocity analytical ultracentrifugation. Left panels: sedimentation distribution plots obtained with the program SEDFIT (Schuck, 2000); right panels: binding isotherms extracted from the peak areas in the left panel. The complex is underlined in blue; free Spy and free client protein are shown in red and black, respectively. In 40 mM HEPES (pH 7.5), 100 mM sodium chloride, either 30 μM Spy dimer was titrated with Im7A3W (A) or 40 μM Spy dimer was titrated with Im7A3 (B) or Im7A3W75F (C). All binding isotherms (blue, right panels) were fit with a quadratic equation to obtain the stoichiometry of the complex. The molecular mass of the complex was calculated directly from the sedimentation distribution plot (left) via SEDFIT. (A) The stoichiometry of the Spy dimer:Im7A3W complex is 0.8 ± 0.1 and the molecular mass is 41.0 ± 1.7 kDa. (B) The stoichiometry of the Spy dimer:Im7A3 complex is 0.8 ± 0.2 and the molecular mass is 40.9 ± 3.6 kDa. (C) The stoichiometry of the Spy dimer:Im7A3W75F complex is 0.8 ± 0.1 and the molecular mass is 42.3 ± 1.4 kDa. Note that the molecular mass of the Spy dimer alone is 31.9 kDa and that of Im7A3 or Im7A3W75F alone is 9.7 kDa. Like Im7A3, Im7A3W and Im7A3W75F bind the Spy dimer in a 1:1 ratio, indicating that the tryptophan insertion or deletion at position 40 or 75 in Im7A3 had no influence on the oligomeric state of the complex.
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12. Figure S3
Transient Kinetics of Spy-Im7A3W Interaction Monitored by Stopped-Flow Fluorescence to Determine Binding (kon) and Release (koff) Rate Constants, Related to Figures 1, 2, and 3.
(A–E) To determine if Spy-Im7A3W binding is electrostatically enhanced, we monitored binding (Left) and release (Right) at various ionic strengths (as indicated on the top left of each panel) using a stopped-flow. Left:  μM (A), 0.125 μM (B), 0.25 μM (C), 0.5 μM (D), and 1.5 μM (E) of Im7A3W were mixed with increasing concentrations of Spy to record binding transients. These fluorescence transients caused by Spy interacting with Im7A3W at low ionic strengths (< 100 mM) were monophasic and were therefore fitted with a single exponential function (A, B). However, at high ionic strengths (≥120 mM), the binding transients appeared to be biphasic as the fast, major increase in fluorescence was followed by a slower increase (C, D, and E). Hence, we fit the transients at higher ionic strength to two exponentials. The fluorescence of free Im7A3W is shown as a reference. Whereas the observed rate constant (kobs) derived from the exponential fits of the fast, major phase at all ionic strength followed a linear concentration dependence (see Figure 1B), the slower phase appeared invariant with Spy concentration (H – J). Right: binding competition experiments to determine the release rate constant (koff):  μM (A), 0.125 μM (B), 0.25 μM (C), 0.5 μM (D), and 1.5 μM (E) of Im7A3W in complex with wild-type Spy (0.25 μM (A), 0.5 μM (B), 2 μM (C), 8 μM (D), and 24 μM (E)) was mixed with the tryptophan-free, unfolded Im7 variant Im7A3W75F (6.25 μM (A), 25 μM (B), 50 μM (C), 300 μM (D), and 900 μM (E)). All traces revealed a small second phase and had to be fit to a double exponential function. Fluorescence of free Im7A3W (bottom line) as well as in complex with Spy (top line) are shown as references.
(F and G) To investigate the effect of hydrophobic interactions on the Spy-Im7A3W interaction kinetics, we monitored binding (left) and release (right) of Im7A3W from Spy variants with increased hydrophobicity. Left: 0.25 μM Im7A3W was mixed with increasing amounts of (F) SpyH96L, or (G) SpyQ100L. Traces for SpyH96L were fit with a single exponential function, whereas SpyQ100L had to be fitted with three exponentials. The fluorescence of free Im7A3W is shown as a reference. Derived observed rate constants (kobs) can be found in Figure 3 and panel L and M. Right: binding competition experiments in which 0.25 μM Im7A3W in complex with the respective Spy variant (2 μM SpyH96L (F), 0.5 μM SpyQ100L (G)) was mixed with the tryptophan-free, unfolded Im7 variant Im7A3W75F (75 μM (F), and 100 μM (G)). All traces revealed a small second phase and had to be fit to a double exponential function. Fluorescence of free Im7A3W (bottom line) as well as in complex with Spy (top line) are shown as references.
(H–M) Observed rate constant of the slow phases observed for binding plotted as a function of Spy concentration for Im7A3W mixed with SpyWT (at ionic strength ≥ 0.12 M: (H) 0.12 M, (I) 0.22 M, and (J) 0.32 M) and SpyQ100L (second (L) and third (M) phase). As kobs is invariant with the Spy concentration, presumably these phases correspond to unimolecular steps that occur either before or after binding of Im7A3W to Spy. However, these phases contributed less than 10% to the total amplitude, and as a result kobs cannot be determined very accurately.
All experiments were performed in 40 mM HEPES (pH 7.5), and various sodium chloride concentrations at 22°C. Rate constants can be found in Table S1. Kinetic traces are averages of four replicates. The kobs of individual replicates were plotted to illustrate the variability of the results.
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13. Figure S4
Binding of Clients to Spy Slows Down with Increasing Salt Concentrations, Related to Figure 2
Complex formation kinetics between Spy and its clients casein (top) and carboxymethylated α-lactalbumin (bottom) was monitored by stopped-flow fluorescence.
(A) Representative raw transients for 0.25 μM casein (top) or 0.5 μM carboxymethylated α-lactalbumin (bottom) mixed with increasing concentrations of Spy at an ionic strength of 0.72 M (casein ) or 0.17 M (α-Lactalbumin) in the stopped-flow fluorimeter. Upon mixing with Spy, two phases were observed for both casein and carboxymethylated α-lactalbumin, and therefore traces were fit with a double exponential function to obtain observed rate constants (kobs). Whereas the kobs of the fast phase increased linearly with Spy concentration for both client proteins (see B), the second phase did not change (see C). The kinetic traces are averages of four replicates. Fluorescence of free casein and α-lactalbumin (bottom line in each plot) are shown as references. Note that free α-lactalbumin showed a fluorescence change upon dilution with buffer which was fit with a single exponential (kobs = 14 s−1). This phase also appeared when mixed with Spy (see C bottom).
(B) kobs of the bimolecular step (fast phase, see A) of Spy-client interaction were plotted as a function of Spy dimer concentration to determine the binding rate constant (kon) at increasing ionic strengths (I) and 22°C. Top: 0.25 μM casein was mixed with increasing concentrations of SpyWT at an ionic strength of 0.12 to 1.02 M. Bottom: 0.125 μM (I = 0.12 M), 0.5 μM (I = 0.17 M), 1 μM (I = 0.22 M, 0.27 M) of carboxymethylated α-Lactalbumin was mixed with increasing concentrations of SpyWT. A linear fit of kobs as a function of Spy concentration yielded kon from the slope. At an ionic strength of 0.12 M, Spy binds to casein with a kon of 2.2 ± 0.2 × 108 M−1 s−1 and to α-lactalbumin with a kon of 2.6 ± 0.4 × 108 M−1 s−1, which is comparable to what was observed for the interaction of Spy with Im7A3W (Figure 2 and Table S1). The ionic strength was adjusted with sodium chloride in 40 mM HEPES (pH 7.5). The kobs of four independent data points for each Spy concentration were plotted to show the experimental error.
(C) Representative kobs of the slow phases observed for binding of casein (top, I = 0.72 M) and α-Lactalbumin (bottom, I = 0.17 M) plotted as a function of SpyWT concentration. As kobs is invariant to the Spy concentration in both cases, presumably these phases correspond to unimolecular steps that occur either before or after binding of client to Spy. The kobs of four independent data points per Spy concentration were plotted to show the experimental error.
(D) The binding rate constant kon of Spy-client interaction as a function of ionic strength for casein (top) and α-lactalbumin (bottom) was derived from the slope of the linear fits of the observed rate constants (see B). Note that the color of each kon was chosen such that it matches the corresponding ionic strength in panel B. As observed for Im7A3W, the kon for both clients binding to Spy drops exponentially with an increase in ionic strength. Errors are propagated fitting standard errors.
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14. Figure S5
Representative ITC Titrations for the Different Im7 and Spy Variants Used, Related to Figures 4 and 5
(A) RNaseA (3 mM in the syringe) does not bind to Im7A3W (0.3 mM in the cell) as determined by ITC.
(B–D) Spy-Im7 interaction is an entropy driven process as measured via ITC, indicating that desolvation of hydrophobic surface in the binding interface is a major contributor to complex stability. Representative binding isotherms at 22°C and an ionic strength of 0.12 M. SpyWT + Im7A3 (B); Spy H96L + Im7A3W (C); Spy Q100L + Im7A3W (D).
(E and F) Representative binding isotherms of Im7A3W (E) and Im7WT (F) titrated with SpyWT at 4°C and an ionic strength of M. Integrated thermograms (bottom graphs) are fit to a single-site-binding-model.
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15. Figure S6
Increased Affinity of Im7A3W for Spy Binding Compared to Im7A3 Is Due to a Decrease in Binding Enthalpy, Related to Figure 4
(A–D) The difference in Kd between Im7A3 and Im7A3W binding to Spy may indicate that the tryptophan substitution may stabilize the complex through additional hydrophobic interactions. To quantify a potential difference in hydrophobic surface area involved in complex formation between Im7A3 and Im7A3W with Spy, we determined the enthalpy (ΔH) (A), entropy (ΔS) (B), and Kd (C) of Im7A3 (black) and Im7A3W (red) titrated with wild-type Spy as a function of temperature via ITC. Spy binding to Im7A3 is more endothermic than Spy binding to Im7A3W (A). The more endothermic binding reaction of Im7A3 to Spy may be a result of the partially positive charge of H40 interfering with binding. In contrast, the reaction entropy is not affected by the H40W substitution (B), indicating that the H40W mutation does not significantly increase hydrophobic interactions in the Spy-Im7A3W complex compared to Im7A3. To confirm this, the heat capacity (ΔCp) for complex formation was determined by fitting ΔH versus temperature to a line (A). A ΔCp of −415.5 ± 7.8 and −426.3 ± 8.3 cal mol−1 K−1 for the interaction of wild-type Spy with either Im7A3 or Im7A3W, respectively, was determined from the slope of this linear fit. As these values are, within error, identical, we conclude that the increased affinity of Spy for Im7A3W compared to Im7A3 does not result from an increased hydrophobic stabilization of the complex. (D) Enthalpy-entropy compensation allows Spy-Im7 complex formation throughout a broad temperature range. Spy interaction is shown with Im7A3W (red dots) and Im7A3 (black dots) in 40 mM HEPES (pH 7.5), 100 mM sodium chloride at various temperatures. Three to four independent data points per temperature were plotted to illustrate the variability of the results.
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16. Figure S7
Natively Folded Im7 Is Released 13-Fold Faster than the Unfolded State, Related to Figure 6
(A and B) Binding transients for  μM Im7A3W (A) or 2.5 μM Im7WT (B) mixed with increasing concentrations of SpyWT. Whereas binding of Im7A3W can be observed and appears to be biphasic, binding of Im7WT is too fast and therefore happens in the dead time of the instrument (indicated as “Burst phase”). The small, slow increase in fluorescence in (B) results from partial unfolding of Im7WT due to a higher affinity of Spy for the intermediate folding state of Im7 (Stull et al., 2016). Fluorescence of free Im7 (bottom line) is shown as a reference.
(C) Fluorescence change with increasing Spy concentration during the burst phase in (B), indicative of very rapid binding of Spy to the native state of Im7WT occurring within the dead time of the instrument. Fit with a quadratic equation, a complex stoichiometry of one Im7WT to one Spy dimer and an estimate of the affinity of Spy binding to the native state of Im7 was obtained (0.05 ± 0.06 μM, see Table S1). The initial fluorescence of four replicates per Spy concentration was plotted to illustrate the variability of the results.
(D and E) Single-mix (D) and double-mix (E) binding competition experiments to determine the release rate constant of natively folded Im7. (D) 2.5 μM Im7WT in complex with 4 μM SpyWT was mixed with 50 μM Im7A3W75F and resulted in a double-exponential decay with observed rate constants of 131 ± 7 s−1 (major phase) and 6.4 ± 1.5 s−1 (minor phase). To determine which phase corresponds to the release rate constant of natively folded Im7, double-mixing experiments were performed (E). Complex formation between Spy and Im7WT was allowed to proceed for 6 ms, and was then chased with 50 μM of Im7A3W75F, resulting in only a single phase with an amplitude and observed rate constant comparable to the major phase obtained by the single-mixing experiment (141 ± 4 s−1 compared to 131 ± 7 s−1 in the single mixing experiment). This indicates that the major phase represents release of Im7 from Spy, and the second phase is caused by unfolding or refolding of a subpopulation of partially folded Im7 after release from Spy.
(F and G) Single-mix (F) and double-mix (G) binding competition experiment to determine the release rate constant of unfolded Im7. (F) 0.25 μM Im7A3W in complex with 0.5 μM SpyWT was mixed with 25 μM Im7A3W75F. As two phases were observed, the data were fit with a double exponential, which yielded observed rate constants of 9.8 ± 0.2 s−1 and 0.6 ± 0.1 s−1. To determine which of the two phases corresponds to the release rate constant of unfolded Im7, double-mixing experiments were performed (G), in which complex formation between Spy and Im7 was allowed to proceed for 10 ms. The reaction was then chased with 25 μM of Im7A3W75F, resulting in only a single phase with an amplitude and observed rate constant comparable to the major phase obtained by the single-mix experiment (9.8 ± 0.2 s−1 in both chases).
Fluorescence of free Im7 (bottom line) as well as in complex with Spy (top line) are shown as references (D, E, F, and G). All experiments were performed in 40 mM HEPES (pH 7.5), 100 mM sodium chloride concentrations at 4°C. Rate constants can also be found in Table S1. Kinetic traces are averages of four replicates.
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