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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

A free energy diagram of h-apoMb at pH 6.0 and 8°C as a function of α-value (i.e., the change in the solvent accessible surface area relative to N).
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pone.0134238.g005: A free energy diagram of h-apoMb at pH 6.0 and 8°C as a function of α-value (i.e., the change in the solvent accessible surface area relative to N).

Mentions: In this study, we showed that the same set of kinetic barriers were encountered in the formation of the kinetic intermediate (M) and the corresponding equilibrium state (Meq) for urea-induced folding and unfolding. In particular, we showed, using CF and SF methods (Fig 4), that M shifts from a transient intermediate to a well populated equilibrium state as the urea concentration is increased. In the refolding reaction, M transiently accumulated during refolding at pH 6.0 and 0 M urea, and formation of M was continuously observed in the refolding up to 2.2 M urea as long as the fast phase λ1 had measurable amplitude. In contrast, the formation of N was observed only at <~1.6 M urea due to the low stability of N (the free energy difference between N and M is ~1.0 kcal/mol at pH 6.0 and 0 M urea; see Table 1). It follows that M predominantly accumulates as an equilibrium intermediate between 0.7 and 2.0 M urea, a urea concentration range in which N is no longer populated predominantly at equilibrium. More intuitively, at low urea concentrations, M was preferentially formed over I because kIM >> kMI, and N', which is readily converted into N, was preferentially formed over M because kMN' is larger than the apparent unfolding rate constant (λ2 ≈ kNN'/(kNN' + kN'N) × kN'M); this leads to the conversion of U to N with transient accumulation of M as a kinetic intermediate. In contrast, at 0.7–2 M urea, M was preferentially formed not only over N' because λ2 was larger than kMN' but also over I because kIM >> kMI; thus, M is stably populated as an equilibrium state, which is equivalent to Meq. Therefore, M and Meq are formed by overcoming the same set of kinetic barriers, consistent with a single molecular species. Furthermore, the stability of N relative to M determines whether M transiently accumulates during refolding under strongly native conditions (~0 M urea) or is populated at equilibrium under moderately denaturing conditions (0.7–2.0 M urea). Moreover, the matching urea dependence of λ2 and λ2' indicates that M and Meq is converted into N via the same kinetics process, as previously reported for the pH-induced folding of sw-apoMb [46]. Quantitative modeling also revealed that M and Meq had similar thermodynamic stabilities and fluorescence intensities (Fig 3A, Tables 1 and 2). This is supported by the shared transition region of F0R2 and Feq at ~1.6 M urea because the urea dependence of F0R2 represented the pre-equilibrium unfolding of M. The energetics of the (un)folding reaction are schematically illustrated by a free energy diagram as a function of α-value, a measure of the change in solvent-accessible surface area occurring during folding. Fig 5 shows that M is one of the most stable species at 1–2 M urea, which leads to the accumulation of M as an equilibrium intermediate. This implies that regions that are more robust to perturbation by urea form at an earlier stage of folding, which further supports the validity of the use of equilibrium intermediates as counterparts for kinetic intermediates. It should be noted that formation of the overall native-like structure (i.e., folding of the C-, D-, E- and part of the B-helix regions that are less stable than the A-, G-, and H-helix regions) would occur during the M → N conversion (phase 2) considering the similarity in the rate-limiting step of folding between h-apoMb and sw-apoMb.


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)

A free energy diagram of h-apoMb at pH 6.0 and 8°C as a function of α-value (i.e., the change in the solvent accessible surface area relative to N).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134238.g005: A free energy diagram of h-apoMb at pH 6.0 and 8°C as a function of α-value (i.e., the change in the solvent accessible surface area relative to N).
Mentions: In this study, we showed that the same set of kinetic barriers were encountered in the formation of the kinetic intermediate (M) and the corresponding equilibrium state (Meq) for urea-induced folding and unfolding. In particular, we showed, using CF and SF methods (Fig 4), that M shifts from a transient intermediate to a well populated equilibrium state as the urea concentration is increased. In the refolding reaction, M transiently accumulated during refolding at pH 6.0 and 0 M urea, and formation of M was continuously observed in the refolding up to 2.2 M urea as long as the fast phase λ1 had measurable amplitude. In contrast, the formation of N was observed only at <~1.6 M urea due to the low stability of N (the free energy difference between N and M is ~1.0 kcal/mol at pH 6.0 and 0 M urea; see Table 1). It follows that M predominantly accumulates as an equilibrium intermediate between 0.7 and 2.0 M urea, a urea concentration range in which N is no longer populated predominantly at equilibrium. More intuitively, at low urea concentrations, M was preferentially formed over I because kIM >> kMI, and N', which is readily converted into N, was preferentially formed over M because kMN' is larger than the apparent unfolding rate constant (λ2 ≈ kNN'/(kNN' + kN'N) × kN'M); this leads to the conversion of U to N with transient accumulation of M as a kinetic intermediate. In contrast, at 0.7–2 M urea, M was preferentially formed not only over N' because λ2 was larger than kMN' but also over I because kIM >> kMI; thus, M is stably populated as an equilibrium state, which is equivalent to Meq. Therefore, M and Meq are formed by overcoming the same set of kinetic barriers, consistent with a single molecular species. Furthermore, the stability of N relative to M determines whether M transiently accumulates during refolding under strongly native conditions (~0 M urea) or is populated at equilibrium under moderately denaturing conditions (0.7–2.0 M urea). Moreover, the matching urea dependence of λ2 and λ2' indicates that M and Meq is converted into N via the same kinetics process, as previously reported for the pH-induced folding of sw-apoMb [46]. Quantitative modeling also revealed that M and Meq had similar thermodynamic stabilities and fluorescence intensities (Fig 3A, Tables 1 and 2). This is supported by the shared transition region of F0R2 and Feq at ~1.6 M urea because the urea dependence of F0R2 represented the pre-equilibrium unfolding of M. The energetics of the (un)folding reaction are schematically illustrated by a free energy diagram as a function of α-value, a measure of the change in solvent-accessible surface area occurring during folding. Fig 5 shows that M is one of the most stable species at 1–2 M urea, which leads to the accumulation of M as an equilibrium intermediate. This implies that regions that are more robust to perturbation by urea form at an earlier stage of folding, which further supports the validity of the use of equilibrium intermediates as counterparts for kinetic intermediates. It should be noted that formation of the overall native-like structure (i.e., folding of the C-, D-, E- and part of the B-helix regions that are less stable than the A-, G-, and H-helix regions) would occur during the M → N conversion (phase 2) considering the similarity in the rate-limiting step of folding between h-apoMb and sw-apoMb.

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