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Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments.

Mizukami T, Abe Y, Maki K - PLoS ONE (2015)

Bottom Line: A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism.Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates.An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Science, Nagoya University, Nagoya, Aichi, Japan.

ABSTRACT
In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0-2.2 M) than the formation of the native state (0-1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7-2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.

No MeSH data available.


Related in: MedlinePlus

Urea-dependence of the rate constants and the cumulative amplitudes of refolding and unfolding of h-apoMb and the results obtained by the quantitative modeling.(A) Urea-dependence of the rate constants of refolding/unfolding reactions obtained in CF and SF experiments (circles): λ1 (green), λ2 (blue for refolding and red for unfolding), λ2' (cyan), and λ3 (orange). Black and colored solid lines show the rate constants (λ1, λ2, and λ3) and elementary rate constants, respectively, predicted by the quantitative modeling (color codes for the elementary rate constants are shown). (B) The cumulative amplitudes obtained in the CF and SF experiments (circles). Open circles: F0R1 (green) and F0R2 (blue) for refolding, and F0U1 (orange) and F0U2 (red) for unfolding. Filled circles: Feq obtained by refolding (blue) and unfolding (red). The solid lines represent Feq (black), F0R1 (green), F0R2 (blue), F0U1 (orange), and F0U2 (red) reproduced by quantitative modeling. Here, the cumulative amplitudes are defined as F0R2 = Feq + F2 and F0R1 = Feq + F2 + F1 for the refolding reaction initiated at pH 2.0, and F0U2 = Feq + F2 and F0U1 = Feq + F2 + F3 for the unfolding reaction, where Fi is the amplitude of the i-th phase of Eq 1 and Feq represents the fluorescence intensity at equilibrium. Dashed lines represent the fluorescence intensities of U (red), I (orange), M (green), N' (cyan), and N (blue) predicted by the quantitative modeling. (C) The fractions of each state reproduced by the quantitative modeling. The solid lines represent the reproduced fractions of U (red), I (orange), M (green), N' (cyan) and N (blue), while the dotted lines in pale colors represent the fractional Ueq (red), Meq (green), and Neq (blue) obtained by the equilibrium unfolding experiments.
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pone.0134238.g004: Urea-dependence of the rate constants and the cumulative amplitudes of refolding and unfolding of h-apoMb and the results obtained by the quantitative modeling.(A) Urea-dependence of the rate constants of refolding/unfolding reactions obtained in CF and SF experiments (circles): λ1 (green), λ2 (blue for refolding and red for unfolding), λ2' (cyan), and λ3 (orange). Black and colored solid lines show the rate constants (λ1, λ2, and λ3) and elementary rate constants, respectively, predicted by the quantitative modeling (color codes for the elementary rate constants are shown). (B) The cumulative amplitudes obtained in the CF and SF experiments (circles). Open circles: F0R1 (green) and F0R2 (blue) for refolding, and F0U1 (orange) and F0U2 (red) for unfolding. Filled circles: Feq obtained by refolding (blue) and unfolding (red). The solid lines represent Feq (black), F0R1 (green), F0R2 (blue), F0U1 (orange), and F0U2 (red) reproduced by quantitative modeling. Here, the cumulative amplitudes are defined as F0R2 = Feq + F2 and F0R1 = Feq + F2 + F1 for the refolding reaction initiated at pH 2.0, and F0U2 = Feq + F2 and F0U1 = Feq + F2 + F3 for the unfolding reaction, where Fi is the amplitude of the i-th phase of Eq 1 and Feq represents the fluorescence intensity at equilibrium. Dashed lines represent the fluorescence intensities of U (red), I (orange), M (green), N' (cyan), and N (blue) predicted by the quantitative modeling. (C) The fractions of each state reproduced by the quantitative modeling. The solid lines represent the reproduced fractions of U (red), I (orange), M (green), N' (cyan) and N (blue), while the dotted lines in pale colors represent the fractional Ueq (red), Meq (green), and Neq (blue) obtained by the equilibrium unfolding experiments.

Mentions: Fig 4A shows the urea-dependence of the rate constants for folding and unfolding (chevron plot). For refolding initiated at pH 2.0, the slower decreasing phase (phase 2; λ2 ≈ 2–5 s-1) corresponds to the rate-limiting step of the overall folding reaction [48,63]. This is consistent with the finding that the rate constant of the slower unfolding phase coincides with λ2 at matching urea concentrations. The only phase observed in refolding initiated at 0.8 M urea and pH 6.0 (phase 2'; λ2' ≈ 2–5 s-1) also overlaps with λ2 under matching conditions (Fig 4A), indicating that Meq and the kinetic intermediate overcome a common rate-limiting step in the urea-induced folding process. On the other hand, the faster increasing phase in refolding initiated at pH 2.0 (phase 1; λ1 ≈ (0.4–1.2) × 104 s-1) corresponds to the formation of a kinetic intermediate (M). The refolding limb exhibits a slight curvature (rollover) at low urea concentrations (~0.8 M urea), suggesting accumulation of an additional intermediate (I) within the dead-time of the CF measurements. The faster of the two unfolding phases observed at 3–4 M urea (phase 3; λ3 ≈ 4 × 103 s-1) is distinct from λ1, indicating the formation of a kinetic unfolding intermediate (N'). The accumulation of N' also accounts for the rollover of the unfolding limb of λ2 at ~3.5 M urea. Transient accumulation of a kinetic unfolding intermediate has previously been reported during the pH-induced unfolding of sw-apoMb [52,64]. Native-like unfolding intermediates are assumed to account for the rollover of the unfolding limb for some proteins [65–67]. However, in contrast to N' (and the reported unfolding intermediate of sw-apoMb), these unfolding intermediates have negligible population during refolding and unfolding because they are always less stable than others under these conditions. The structural and kinetic properties of N' remain to be elucidated.


Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments.

Mizukami T, Abe Y, Maki K - PLoS ONE (2015)

Urea-dependence of the rate constants and the cumulative amplitudes of refolding and unfolding of h-apoMb and the results obtained by the quantitative modeling.(A) Urea-dependence of the rate constants of refolding/unfolding reactions obtained in CF and SF experiments (circles): λ1 (green), λ2 (blue for refolding and red for unfolding), λ2' (cyan), and λ3 (orange). Black and colored solid lines show the rate constants (λ1, λ2, and λ3) and elementary rate constants, respectively, predicted by the quantitative modeling (color codes for the elementary rate constants are shown). (B) The cumulative amplitudes obtained in the CF and SF experiments (circles). Open circles: F0R1 (green) and F0R2 (blue) for refolding, and F0U1 (orange) and F0U2 (red) for unfolding. Filled circles: Feq obtained by refolding (blue) and unfolding (red). The solid lines represent Feq (black), F0R1 (green), F0R2 (blue), F0U1 (orange), and F0U2 (red) reproduced by quantitative modeling. Here, the cumulative amplitudes are defined as F0R2 = Feq + F2 and F0R1 = Feq + F2 + F1 for the refolding reaction initiated at pH 2.0, and F0U2 = Feq + F2 and F0U1 = Feq + F2 + F3 for the unfolding reaction, where Fi is the amplitude of the i-th phase of Eq 1 and Feq represents the fluorescence intensity at equilibrium. Dashed lines represent the fluorescence intensities of U (red), I (orange), M (green), N' (cyan), and N (blue) predicted by the quantitative modeling. (C) The fractions of each state reproduced by the quantitative modeling. The solid lines represent the reproduced fractions of U (red), I (orange), M (green), N' (cyan) and N (blue), while the dotted lines in pale colors represent the fractional Ueq (red), Meq (green), and Neq (blue) obtained by the equilibrium unfolding experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526358&req=5

pone.0134238.g004: Urea-dependence of the rate constants and the cumulative amplitudes of refolding and unfolding of h-apoMb and the results obtained by the quantitative modeling.(A) Urea-dependence of the rate constants of refolding/unfolding reactions obtained in CF and SF experiments (circles): λ1 (green), λ2 (blue for refolding and red for unfolding), λ2' (cyan), and λ3 (orange). Black and colored solid lines show the rate constants (λ1, λ2, and λ3) and elementary rate constants, respectively, predicted by the quantitative modeling (color codes for the elementary rate constants are shown). (B) The cumulative amplitudes obtained in the CF and SF experiments (circles). Open circles: F0R1 (green) and F0R2 (blue) for refolding, and F0U1 (orange) and F0U2 (red) for unfolding. Filled circles: Feq obtained by refolding (blue) and unfolding (red). The solid lines represent Feq (black), F0R1 (green), F0R2 (blue), F0U1 (orange), and F0U2 (red) reproduced by quantitative modeling. Here, the cumulative amplitudes are defined as F0R2 = Feq + F2 and F0R1 = Feq + F2 + F1 for the refolding reaction initiated at pH 2.0, and F0U2 = Feq + F2 and F0U1 = Feq + F2 + F3 for the unfolding reaction, where Fi is the amplitude of the i-th phase of Eq 1 and Feq represents the fluorescence intensity at equilibrium. Dashed lines represent the fluorescence intensities of U (red), I (orange), M (green), N' (cyan), and N (blue) predicted by the quantitative modeling. (C) The fractions of each state reproduced by the quantitative modeling. The solid lines represent the reproduced fractions of U (red), I (orange), M (green), N' (cyan) and N (blue), while the dotted lines in pale colors represent the fractional Ueq (red), Meq (green), and Neq (blue) obtained by the equilibrium unfolding experiments.
Mentions: Fig 4A shows the urea-dependence of the rate constants for folding and unfolding (chevron plot). For refolding initiated at pH 2.0, the slower decreasing phase (phase 2; λ2 ≈ 2–5 s-1) corresponds to the rate-limiting step of the overall folding reaction [48,63]. This is consistent with the finding that the rate constant of the slower unfolding phase coincides with λ2 at matching urea concentrations. The only phase observed in refolding initiated at 0.8 M urea and pH 6.0 (phase 2'; λ2' ≈ 2–5 s-1) also overlaps with λ2 under matching conditions (Fig 4A), indicating that Meq and the kinetic intermediate overcome a common rate-limiting step in the urea-induced folding process. On the other hand, the faster increasing phase in refolding initiated at pH 2.0 (phase 1; λ1 ≈ (0.4–1.2) × 104 s-1) corresponds to the formation of a kinetic intermediate (M). The refolding limb exhibits a slight curvature (rollover) at low urea concentrations (~0.8 M urea), suggesting accumulation of an additional intermediate (I) within the dead-time of the CF measurements. The faster of the two unfolding phases observed at 3–4 M urea (phase 3; λ3 ≈ 4 × 103 s-1) is distinct from λ1, indicating the formation of a kinetic unfolding intermediate (N'). The accumulation of N' also accounts for the rollover of the unfolding limb of λ2 at ~3.5 M urea. Transient accumulation of a kinetic unfolding intermediate has previously been reported during the pH-induced unfolding of sw-apoMb [52,64]. Native-like unfolding intermediates are assumed to account for the rollover of the unfolding limb for some proteins [65–67]. However, in contrast to N' (and the reported unfolding intermediate of sw-apoMb), these unfolding intermediates have negligible population during refolding and unfolding because they are always less stable than others under these conditions. The structural and kinetic properties of N' remain to be elucidated.

Bottom Line: A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism.Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates.An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Science, Nagoya University, Nagoya, Aichi, Japan.

ABSTRACT
In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0-2.2 M) than the formation of the native state (0-1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7-2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.

No MeSH data available.


Related in: MedlinePlus