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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

Ribbon diagram of horse skeletal muscle myoglobin on a crystallographic structure (PDB: 1AZI).The heme group (light gray) and two tryptophanyl side chains (black) are shown in the ball-and-stick model. The native state of myoglobin contains eight helices (A–H), whereas the native state of h-apoMb contains seven helices (A–E, G, and H; black and gray). The intermediate of apoMb exhibits a helical structure in the A-, G-, and H-helix regions (black). The F-helix is shown in light gray. The figure was prepared using PyMol (DeLano Scientific).
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pone.0134238.g001: Ribbon diagram of horse skeletal muscle myoglobin on a crystallographic structure (PDB: 1AZI).The heme group (light gray) and two tryptophanyl side chains (black) are shown in the ball-and-stick model. The native state of myoglobin contains eight helices (A–H), whereas the native state of h-apoMb contains seven helices (A–E, G, and H; black and gray). The intermediate of apoMb exhibits a helical structure in the A-, G-, and H-helix regions (black). The F-helix is shown in light gray. The figure was prepared using PyMol (DeLano Scientific).

Mentions: Apomyoglobin (apoMb) is an excellent model protein for investigating these questions because the properties of kinetic and equilibrium intermediates and folding mechanisms have been studied extensively [19,20]. Myoglobin is a heme protein with 153 amino acid residues consisting of eight helices (A–H) that adopt a globin fold [21]. Early studies found that the protein exhibits fully native properties without heme, but is slightly less compact and more disordered than the holo form [22–24]. ApoMb, which lacks a heme group, accumulates equilibrium unfolding intermediates under various moderately denaturing conditions [25–27], including acidic conditions in the presence of salt [28,29]. Equilibrium unfolding experiments on sperm whale apomyoglobin (sw-apoMb) indicated that the pH/urea-induced intermediate was approximated by a single thermodynamic state [30]. In addition, there was no clear transition between the salt-induced and pH-induced intermediates for horse skeletal muscle apomyoglobin (h-apoMb), which suggests that a detectable thermodynamic barrier does not exist between them [28]. Further structural characterization revealed that the intermediate at pH 4 (hereafter known as "the pH 4 intermediate") is compact with a structural core consisting of a native-like helical structure in the A-, G-, H-, and part of the B-helix regions [31–34]; however, under physiological conditions, it forms a compact, native structure similar to that of native myoglobin, with the F-helix, the N-terminus of the G-helix, and some loops unstructured (Fig 1) [35,36]. In contrast, the acid unfolded state at pH 2.3 was reported to be highly flexible with residual helical structures in the A-, H-, and D- to N-terminus of the E-helix regions in dynamic equilibrium with the unfolded state [33,37,38], which is consistent with previous studies of peptide fragments [39–41]. In another study, the addition of 8 M urea to the solution at pH 2.3 further denatured the protein with virtually no residual structures [42]. The folding kinetics of sw-apoMb was studied by pulsed hydrogen/deuterium (H/D) exchange experiments, and these revealed that helical structure is formed in the A-, G-, H-, and part of the B- helices of the native structure on a millisecond-to-second time scale during the folding reaction at pH 6.1 [6]. The intermediate ensemble has a hydrophobic core surrounded by native-like secondary structures with specific interactions between side-chains [43,44]. Further kinetic studies of folding indicated that sw-apoMb follows a sequential folding pathway [45] and accumulates at least two kinetic intermediates [46,47]. H-apoMb also folds via two kinetic intermediates with a compact size and considerable fraction of helical structure on a submillisecond-to-second time scale [48]. A submillisecond pulsed H/D exchange experiment with sw-apoMb revealed that a compact intermediate is formed with a helical structure in the A-, G-, and H-helix regions within 0.4 ms of the refolding [5]. The effects of protonation and temperature on the denaturation of apoMb and myoglobin were also investigated in previous simulation studies [49–51]. Although the solution conditions in which the equilibrium intermediate accumulates are far from native conditions, i.e., the conditions where kinetic intermediates transiently accumulate, the equilibrium and kinetic states exhibit similar structural properties (e.g., spectroscopic properties and patterns of amide protection in H/D exchange) [5–7]. If they are equivalent, the kinetic and equilibrium intermediates should share a common kinetic mechanism of formation. Although the rate-limiting step of the pH-induced folding reactions of sw-apoMb from kinetic and equilibrium intermediates was found to be similar by comparing their refolding kinetics initiated at pH 2.2, 3.4, and 4.2 [46], conventional kinetic measurements were too slow for a direct comparison of the kinetics of formation of transient and equilibrium intermediates.


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)

Ribbon diagram of horse skeletal muscle myoglobin on a crystallographic structure (PDB: 1AZI).The heme group (light gray) and two tryptophanyl side chains (black) are shown in the ball-and-stick model. The native state of myoglobin contains eight helices (A–H), whereas the native state of h-apoMb contains seven helices (A–E, G, and H; black and gray). The intermediate of apoMb exhibits a helical structure in the A-, G-, and H-helix regions (black). The F-helix is shown in light gray. The figure was prepared using PyMol (DeLano Scientific).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134238.g001: Ribbon diagram of horse skeletal muscle myoglobin on a crystallographic structure (PDB: 1AZI).The heme group (light gray) and two tryptophanyl side chains (black) are shown in the ball-and-stick model. The native state of myoglobin contains eight helices (A–H), whereas the native state of h-apoMb contains seven helices (A–E, G, and H; black and gray). The intermediate of apoMb exhibits a helical structure in the A-, G-, and H-helix regions (black). The F-helix is shown in light gray. The figure was prepared using PyMol (DeLano Scientific).
Mentions: Apomyoglobin (apoMb) is an excellent model protein for investigating these questions because the properties of kinetic and equilibrium intermediates and folding mechanisms have been studied extensively [19,20]. Myoglobin is a heme protein with 153 amino acid residues consisting of eight helices (A–H) that adopt a globin fold [21]. Early studies found that the protein exhibits fully native properties without heme, but is slightly less compact and more disordered than the holo form [22–24]. ApoMb, which lacks a heme group, accumulates equilibrium unfolding intermediates under various moderately denaturing conditions [25–27], including acidic conditions in the presence of salt [28,29]. Equilibrium unfolding experiments on sperm whale apomyoglobin (sw-apoMb) indicated that the pH/urea-induced intermediate was approximated by a single thermodynamic state [30]. In addition, there was no clear transition between the salt-induced and pH-induced intermediates for horse skeletal muscle apomyoglobin (h-apoMb), which suggests that a detectable thermodynamic barrier does not exist between them [28]. Further structural characterization revealed that the intermediate at pH 4 (hereafter known as "the pH 4 intermediate") is compact with a structural core consisting of a native-like helical structure in the A-, G-, H-, and part of the B-helix regions [31–34]; however, under physiological conditions, it forms a compact, native structure similar to that of native myoglobin, with the F-helix, the N-terminus of the G-helix, and some loops unstructured (Fig 1) [35,36]. In contrast, the acid unfolded state at pH 2.3 was reported to be highly flexible with residual helical structures in the A-, H-, and D- to N-terminus of the E-helix regions in dynamic equilibrium with the unfolded state [33,37,38], which is consistent with previous studies of peptide fragments [39–41]. In another study, the addition of 8 M urea to the solution at pH 2.3 further denatured the protein with virtually no residual structures [42]. The folding kinetics of sw-apoMb was studied by pulsed hydrogen/deuterium (H/D) exchange experiments, and these revealed that helical structure is formed in the A-, G-, H-, and part of the B- helices of the native structure on a millisecond-to-second time scale during the folding reaction at pH 6.1 [6]. The intermediate ensemble has a hydrophobic core surrounded by native-like secondary structures with specific interactions between side-chains [43,44]. Further kinetic studies of folding indicated that sw-apoMb follows a sequential folding pathway [45] and accumulates at least two kinetic intermediates [46,47]. H-apoMb also folds via two kinetic intermediates with a compact size and considerable fraction of helical structure on a submillisecond-to-second time scale [48]. A submillisecond pulsed H/D exchange experiment with sw-apoMb revealed that a compact intermediate is formed with a helical structure in the A-, G-, and H-helix regions within 0.4 ms of the refolding [5]. The effects of protonation and temperature on the denaturation of apoMb and myoglobin were also investigated in previous simulation studies [49–51]. Although the solution conditions in which the equilibrium intermediate accumulates are far from native conditions, i.e., the conditions where kinetic intermediates transiently accumulate, the equilibrium and kinetic states exhibit similar structural properties (e.g., spectroscopic properties and patterns of amide protection in H/D exchange) [5–7]. If they are equivalent, the kinetic and equilibrium intermediates should share a common kinetic mechanism of formation. Although the rate-limiting step of the pH-induced folding reactions of sw-apoMb from kinetic and equilibrium intermediates was found to be similar by comparing their refolding kinetics initiated at pH 2.2, 3.4, and 4.2 [46], conventional kinetic measurements were too slow for a direct comparison of the kinetics of formation of transient and equilibrium intermediates.

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