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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

The kinetic traces of refolding and unfolding reactions of h-apoMb.The kinetic traces of (A) refolding and (B) unfolding reactions were measured by CF and SF fluorescence methods at various urea concentrations, pH 6.0, and 8°C. The fluorescence intensity is scaled relative to the fluorescence at pH 2.0. Black and colored lines show the kinetic traces obtained by non-linear least squares fitting and by the kinetic modeling, respectively. Kinetic parameters used to reconstruct kinetic traces are shown in Table C in S1 File (elementary rate constants and kinetic m-values) and Table 2 (intrinsic fluorescence intensity of each structural species).
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pone.0134238.g003: The kinetic traces of refolding and unfolding reactions of h-apoMb.The kinetic traces of (A) refolding and (B) unfolding reactions were measured by CF and SF fluorescence methods at various urea concentrations, pH 6.0, and 8°C. The fluorescence intensity is scaled relative to the fluorescence at pH 2.0. Black and colored lines show the kinetic traces obtained by non-linear least squares fitting and by the kinetic modeling, respectively. Kinetic parameters used to reconstruct kinetic traces are shown in Table C in S1 File (elementary rate constants and kinetic m-values) and Table 2 (intrinsic fluorescence intensity of each structural species).

Mentions: The refolding and unfolding kinetics of h-apoMb was measured by monitoring fluorescence at various urea concentrations, pH 6.0, and 8°C. The refolding reaction at pH 6.0 was initiated by mixing acid-unfolded protein (pH 2.0) with buffer containing appropriate concentrations of urea, whereas the unfolding reaction at pH 6.0 was initiated by mixing native protein (pH 6.0) with buffer containing appropriate concentrations of urea. CF and SF experiments were combined to cover the time course of folding and unfolding over the time range from ~100 μs to minutes. The dead-times of the CF and SF measurements were 102–175μs (depending on the in-house constructed mixers used in the CF experiments) and 5.3 ms, respectively. Representative kinetic traces of the refolding and unfolding reactions are shown in Fig 3. For the refolding reaction, the kinetic traces measured at urea concentrations (≤1.6 M) were fitted to a double-exponential function consisting of a faster rising phase and a slower decreasing phase whereas those at higher urea concentrations were fitted to a rising single-exponential function. The transient increase and subsequent decrease in fluorescence observed in refolding experiments at low urea concentrations reflects the rapid accumulation of a kinetic intermediate (M; see below) followed by conversion into the native state (0.1–1 s). In contrast, the increase in fluorescence without the slower decreasing phase observed at higher urea concentrations indicates that the kinetic intermediate remained populated as an equilibrium intermediate, Meq. The observation indicates that the kinetic intermediate was continuously converted into Meq as the final urea concentration increased, and that both M and Meq were formed by the same kinetic mechanism from the preceding state even under considerably different conditions (i.e., M transiently accumulated even in the absence of urea, whereas Meq was stably populated even at urea concentrations higher than 2 M; see below). In addition to the kinetic phase(s) resolved by the fitting, an unresolved change in fluorescence (burst phase) was observed in the CF measurements from 0 M to 3.0 M urea, indicating accumulation of an additional intermediate, I, within the 100-μs range (see below). We also measured the refolding kinetics initiated at pH 6.0 and 0.8 M urea, where Meq is a major non-native species (Fig 2D), by mixing the protein solution with buffer containing appropriate concentrations of urea in the SF device. In this case, the kinetic trace was fitted to a single-exponential function (0.13–0.53 M urea). This refolding reaction was dominated by the conversion of Meq to Neq. The kinetic traces of the unfolding reaction were fitted to a double-exponential function at intermediate urea concentrations (3.0–4.0 M urea) and a single-exponential function at lower and higher concentrations. The transient enhancement of the fluorescence observed at 3–4 M urea (within 1 ms of unfolding) corresponds to the accumulation of a kinetic unfolding intermediate (N'; see below), which is subsequently converted into the unfolded state (0.01–0.1 s). The faster phase was not observed at low urea concentrations because of its small amplitude. It should be noted that urea was limited to ~4 M in the final conditions for CF unfolding measurement because of the solubility of urea and the 1:1 mixing ratio of the CF device.


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)

The kinetic traces of refolding and unfolding reactions of h-apoMb.The kinetic traces of (A) refolding and (B) unfolding reactions were measured by CF and SF fluorescence methods at various urea concentrations, pH 6.0, and 8°C. The fluorescence intensity is scaled relative to the fluorescence at pH 2.0. Black and colored lines show the kinetic traces obtained by non-linear least squares fitting and by the kinetic modeling, respectively. Kinetic parameters used to reconstruct kinetic traces are shown in Table C in S1 File (elementary rate constants and kinetic m-values) and Table 2 (intrinsic fluorescence intensity of each structural species).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134238.g003: The kinetic traces of refolding and unfolding reactions of h-apoMb.The kinetic traces of (A) refolding and (B) unfolding reactions were measured by CF and SF fluorescence methods at various urea concentrations, pH 6.0, and 8°C. The fluorescence intensity is scaled relative to the fluorescence at pH 2.0. Black and colored lines show the kinetic traces obtained by non-linear least squares fitting and by the kinetic modeling, respectively. Kinetic parameters used to reconstruct kinetic traces are shown in Table C in S1 File (elementary rate constants and kinetic m-values) and Table 2 (intrinsic fluorescence intensity of each structural species).
Mentions: The refolding and unfolding kinetics of h-apoMb was measured by monitoring fluorescence at various urea concentrations, pH 6.0, and 8°C. The refolding reaction at pH 6.0 was initiated by mixing acid-unfolded protein (pH 2.0) with buffer containing appropriate concentrations of urea, whereas the unfolding reaction at pH 6.0 was initiated by mixing native protein (pH 6.0) with buffer containing appropriate concentrations of urea. CF and SF experiments were combined to cover the time course of folding and unfolding over the time range from ~100 μs to minutes. The dead-times of the CF and SF measurements were 102–175μs (depending on the in-house constructed mixers used in the CF experiments) and 5.3 ms, respectively. Representative kinetic traces of the refolding and unfolding reactions are shown in Fig 3. For the refolding reaction, the kinetic traces measured at urea concentrations (≤1.6 M) were fitted to a double-exponential function consisting of a faster rising phase and a slower decreasing phase whereas those at higher urea concentrations were fitted to a rising single-exponential function. The transient increase and subsequent decrease in fluorescence observed in refolding experiments at low urea concentrations reflects the rapid accumulation of a kinetic intermediate (M; see below) followed by conversion into the native state (0.1–1 s). In contrast, the increase in fluorescence without the slower decreasing phase observed at higher urea concentrations indicates that the kinetic intermediate remained populated as an equilibrium intermediate, Meq. The observation indicates that the kinetic intermediate was continuously converted into Meq as the final urea concentration increased, and that both M and Meq were formed by the same kinetic mechanism from the preceding state even under considerably different conditions (i.e., M transiently accumulated even in the absence of urea, whereas Meq was stably populated even at urea concentrations higher than 2 M; see below). In addition to the kinetic phase(s) resolved by the fitting, an unresolved change in fluorescence (burst phase) was observed in the CF measurements from 0 M to 3.0 M urea, indicating accumulation of an additional intermediate, I, within the 100-μs range (see below). We also measured the refolding kinetics initiated at pH 6.0 and 0.8 M urea, where Meq is a major non-native species (Fig 2D), by mixing the protein solution with buffer containing appropriate concentrations of urea in the SF device. In this case, the kinetic trace was fitted to a single-exponential function (0.13–0.53 M urea). This refolding reaction was dominated by the conversion of Meq to Neq. The kinetic traces of the unfolding reaction were fitted to a double-exponential function at intermediate urea concentrations (3.0–4.0 M urea) and a single-exponential function at lower and higher concentrations. The transient enhancement of the fluorescence observed at 3–4 M urea (within 1 ms of unfolding) corresponds to the accumulation of a kinetic unfolding intermediate (N'; see below), which is subsequently converted into the unfolded state (0.01–0.1 s). The faster phase was not observed at low urea concentrations because of its small amplitude. It should be noted that urea was limited to ~4 M in the final conditions for CF unfolding measurement because of the solubility of urea and the 1:1 mixing ratio of the CF device.

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