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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-induced equilibrium unfolding of h-apoMb in 12 mM sodium citrate at pH 6.0 and 8°C.Unfolding transition curves monitored by (A) ellipticity at 222 nm and (B) the tryptophan fluorescence spectrum (310–440 nm). The solid lines are unfolding transition curves predicted by the global fitting of a three-state model (Scheme 1) along with the residuals (upper panel). (A) Broken lines are baselines of Neq (blue), Meq (green), and Ueq (red) predicted by the global fitting. (B) Unfolding curves are coded with different colors. (C) A collection of fluorescence spectra of h-apoMb as a function of urea concentration measured at pH 6.0 and 8°C. Black circles represent an unfolding transition curve monitored by the fluorescence emission at 360 nm. The transition curves in (B) were obtained by transposing data in (C). (D) The fractions of each species as a function of the urea concentration obtained by the equilibrium unfolding experiments. The fractional Neq, Meq, and Ueq are shown in blue, green, and red, respectively.
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pone.0134238.g002: Urea-induced equilibrium unfolding of h-apoMb in 12 mM sodium citrate at pH 6.0 and 8°C.Unfolding transition curves monitored by (A) ellipticity at 222 nm and (B) the tryptophan fluorescence spectrum (310–440 nm). The solid lines are unfolding transition curves predicted by the global fitting of a three-state model (Scheme 1) along with the residuals (upper panel). (A) Broken lines are baselines of Neq (blue), Meq (green), and Ueq (red) predicted by the global fitting. (B) Unfolding curves are coded with different colors. (C) A collection of fluorescence spectra of h-apoMb as a function of urea concentration measured at pH 6.0 and 8°C. Black circles represent an unfolding transition curve monitored by the fluorescence emission at 360 nm. The transition curves in (B) were obtained by transposing data in (C). (D) The fractions of each species as a function of the urea concentration obtained by the equilibrium unfolding experiments. The fractional Neq, Meq, and Ueq are shown in blue, green, and red, respectively.

Mentions: Fig 2 shows the urea-induced equilibrium unfolding of h-apoMb measured by monitoring the changes in ellipticity at 222 nm (Fig 2A) and the Trp fluorescence emission spectra (300–450 nm) upon excitation at 295 nm (Fig 2B and 2C). The measurements were performed in 12 mM sodium citrate at pH 6.0 and 8°C. These unfolding transition curves were similar to those previously reported for sw-apoMb [30,44]. The transition curves monitored by fluorescence at 310–360 nm exhibited two transitions: one at ~1 M urea with an enhancement and the other at ~2 M urea with a decrease. In contrast, those monitored by fluorescence at >370 nm and ellipticity at 222 nm apparently exhibited a single transition at ~1.5 M and ~1 M urea, respectively. They were fitted by non-linear least squares fitting using a global fitting algorithm [9,55,56] to an equilibrium three-state model consisting of the native (Neq), equilibrium intermediate (Meq), and unfolded (Ueq) states as follows:Ueq⇌Meq⇌NeqScheme 1which yielded a set of global parameters (midpoint urea concentrations of Ueq ↔ Meq and Meq ↔ Neq transitions, Cm1, and Cm2, and the corresponding m-values, m1 and m2 for each transition) and local parameters (fluorescence intensities at 300–450 nm and ellipticity at 222 nm for Neq, Meq, and Ueq at 0 M urea, and the slope of the baselines). A linear approximation of the urea-dependence of the baseline as well as the thermodynamic stability of each species was based on the work of Pace and that of Santoro and Bolen [55,56]. The thermodynamic parameters are listed in Table 1. The fractions of the three species were also calculated as a function of urea concentration (Fig 2D). At 0 M urea, h-apoMb consisted of a mixture of Neq (~95%) and Meq (~5%). The fraction of Meq increased from ~5% at 0 M urea, at the expense of the fraction of Neq, until it reached a maximum at 1.3 M urea, which was subsequently followed by a decrease with the increasing fraction of Ueq. We also investigated the stability of h-apoMb vs. temperature to rule out possible cold denaturation [26] at 8°C, the temperature used in this study. Fig A in S1 File shows that ellipticity at 222 nm decreased with temperature down to 4°C, below which cold denaturation occurs. This confirms that h-apoMb maintained its native structure at pH 6.0 and 8°C in the absence of urea.


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-induced equilibrium unfolding of h-apoMb in 12 mM sodium citrate at pH 6.0 and 8°C.Unfolding transition curves monitored by (A) ellipticity at 222 nm and (B) the tryptophan fluorescence spectrum (310–440 nm). The solid lines are unfolding transition curves predicted by the global fitting of a three-state model (Scheme 1) along with the residuals (upper panel). (A) Broken lines are baselines of Neq (blue), Meq (green), and Ueq (red) predicted by the global fitting. (B) Unfolding curves are coded with different colors. (C) A collection of fluorescence spectra of h-apoMb as a function of urea concentration measured at pH 6.0 and 8°C. Black circles represent an unfolding transition curve monitored by the fluorescence emission at 360 nm. The transition curves in (B) were obtained by transposing data in (C). (D) The fractions of each species as a function of the urea concentration obtained by the equilibrium unfolding experiments. The fractional Neq, Meq, and Ueq are shown in blue, green, and red, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134238.g002: Urea-induced equilibrium unfolding of h-apoMb in 12 mM sodium citrate at pH 6.0 and 8°C.Unfolding transition curves monitored by (A) ellipticity at 222 nm and (B) the tryptophan fluorescence spectrum (310–440 nm). The solid lines are unfolding transition curves predicted by the global fitting of a three-state model (Scheme 1) along with the residuals (upper panel). (A) Broken lines are baselines of Neq (blue), Meq (green), and Ueq (red) predicted by the global fitting. (B) Unfolding curves are coded with different colors. (C) A collection of fluorescence spectra of h-apoMb as a function of urea concentration measured at pH 6.0 and 8°C. Black circles represent an unfolding transition curve monitored by the fluorescence emission at 360 nm. The transition curves in (B) were obtained by transposing data in (C). (D) The fractions of each species as a function of the urea concentration obtained by the equilibrium unfolding experiments. The fractional Neq, Meq, and Ueq are shown in blue, green, and red, respectively.
Mentions: Fig 2 shows the urea-induced equilibrium unfolding of h-apoMb measured by monitoring the changes in ellipticity at 222 nm (Fig 2A) and the Trp fluorescence emission spectra (300–450 nm) upon excitation at 295 nm (Fig 2B and 2C). The measurements were performed in 12 mM sodium citrate at pH 6.0 and 8°C. These unfolding transition curves were similar to those previously reported for sw-apoMb [30,44]. The transition curves monitored by fluorescence at 310–360 nm exhibited two transitions: one at ~1 M urea with an enhancement and the other at ~2 M urea with a decrease. In contrast, those monitored by fluorescence at >370 nm and ellipticity at 222 nm apparently exhibited a single transition at ~1.5 M and ~1 M urea, respectively. They were fitted by non-linear least squares fitting using a global fitting algorithm [9,55,56] to an equilibrium three-state model consisting of the native (Neq), equilibrium intermediate (Meq), and unfolded (Ueq) states as follows:Ueq⇌Meq⇌NeqScheme 1which yielded a set of global parameters (midpoint urea concentrations of Ueq ↔ Meq and Meq ↔ Neq transitions, Cm1, and Cm2, and the corresponding m-values, m1 and m2 for each transition) and local parameters (fluorescence intensities at 300–450 nm and ellipticity at 222 nm for Neq, Meq, and Ueq at 0 M urea, and the slope of the baselines). A linear approximation of the urea-dependence of the baseline as well as the thermodynamic stability of each species was based on the work of Pace and that of Santoro and Bolen [55,56]. The thermodynamic parameters are listed in Table 1. The fractions of the three species were also calculated as a function of urea concentration (Fig 2D). At 0 M urea, h-apoMb consisted of a mixture of Neq (~95%) and Meq (~5%). The fraction of Meq increased from ~5% at 0 M urea, at the expense of the fraction of Neq, until it reached a maximum at 1.3 M urea, which was subsequently followed by a decrease with the increasing fraction of Ueq. We also investigated the stability of h-apoMb vs. temperature to rule out possible cold denaturation [26] at 8°C, the temperature used in this study. Fig A in S1 File shows that ellipticity at 222 nm decreased with temperature down to 4°C, below which cold denaturation occurs. This confirms that h-apoMb maintained its native structure at pH 6.0 and 8°C in the absence of urea.

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