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Probing the non-native H helix translocation in apomyoglobin folding intermediates.

Aoto PC, Nishimura C, Dyson HJ, Wright PE - Biochemistry (2014)

Bottom Line: Fluorescence spectra of mutant proteins in which cysteine residues were introduced at several positions in the G and H helices show differential quenching of W14 fluorescence, providing direct evidence of translocation of the H helix relative to helices A and G in both the kinetic and equilibrium intermediates.Formation of an S108C-L135C disulfide prevents H helix translocation in the equilibrium molten globule by locking the G and H helices into their native register.By enforcing nativelike packing of the A, G, and H helices, the disulfide resolves local energetic frustration and facilitates transient docking of the E helix region onto the hydrophobic core but has only a small effect on the refolding rate.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Skaggs Institute of Chemical Biology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

ABSTRACT
Apomyoglobin folds via sequential helical intermediates that are formed by rapid collapse of the A, B, G, and H helix regions. An equilibrium molten globule with a similar structure is formed near pH 4. Previous studies suggested that the folding intermediates are kinetically trapped states in which folding is impeded by non-native packing of the G and H helices. Fluorescence spectra of mutant proteins in which cysteine residues were introduced at several positions in the G and H helices show differential quenching of W14 fluorescence, providing direct evidence of translocation of the H helix relative to helices A and G in both the kinetic and equilibrium intermediates. Förster resonance energy transfer measurements show that a 5-({2-[(acetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid acceptor coupled to K140C (helix H) is closer to Trp14 (helix A) in the equilibrium molten globule than in the native state, by a distance that is consistent with sliding of the H helix in an N-terminal direction by approximately one helical turn. Formation of an S108C-L135C disulfide prevents H helix translocation in the equilibrium molten globule by locking the G and H helices into their native register. By enforcing nativelike packing of the A, G, and H helices, the disulfide resolves local energetic frustration and facilitates transient docking of the E helix region onto the hydrophobic core but has only a small effect on the refolding rate. The apomyoglobin folding landscape is highly rugged, with several energetic bottlenecks that frustrate folding; relief of any one of the major identified bottlenecks is insufficient to speed progression to the transition state.

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Fluorescence decay after a pH jump frompH 2.2 to 6.0 for W7F (black),W7F/M131C (blue), and W7F/L135C (orange). The stopped-flow tracesshow total fluorescence with a 320 nm cutoff filter. The excitationwavelength was 288 nm.
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fig6: Fluorescence decay after a pH jump frompH 2.2 to 6.0 for W7F (black),W7F/M131C (blue), and W7F/L135C (orange). The stopped-flow tracesshow total fluorescence with a 320 nm cutoff filter. The excitationwavelength was 288 nm.

Mentions: Kinetic refolding of apomyoglobin, as measured by stopped-flow CDor fluorescence spectroscopy, occurs in two phases, an initial “burst”phase that is complete within the dead time of the apparatus and aslower (approximately milliseconds) phase that can be monitored directly.The refolding kinetics of the W7F, W7F/M131C, and W7F/L135C mutantswere monitored using stopped-flow fluorescence and CD measurements.The changes in W14 fluorescence emission during kinetic refoldingare shown in Figure 6. The kinetic traces forW7F and W7F/M131C are biphasic, reporting on the rates of the Ia → Ib and Ib → N foldingprocesses,23 and were fit to double-exponentialdecays. For W7F/L135C, the change in fluorescence emission duringthe slow phase (Ib → N) is of extremely small amplitude,because there is little change in fluorescence intensity between themolten globule and native state (Figure 5),and the fast phase dominates the stopped-flow trace (Figure 6). The fitted kinetic parameters are summarizedin Table 2.


Probing the non-native H helix translocation in apomyoglobin folding intermediates.

Aoto PC, Nishimura C, Dyson HJ, Wright PE - Biochemistry (2014)

Fluorescence decay after a pH jump frompH 2.2 to 6.0 for W7F (black),W7F/M131C (blue), and W7F/L135C (orange). The stopped-flow tracesshow total fluorescence with a 320 nm cutoff filter. The excitationwavelength was 288 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4067146&req=5

fig6: Fluorescence decay after a pH jump frompH 2.2 to 6.0 for W7F (black),W7F/M131C (blue), and W7F/L135C (orange). The stopped-flow tracesshow total fluorescence with a 320 nm cutoff filter. The excitationwavelength was 288 nm.
Mentions: Kinetic refolding of apomyoglobin, as measured by stopped-flow CDor fluorescence spectroscopy, occurs in two phases, an initial “burst”phase that is complete within the dead time of the apparatus and aslower (approximately milliseconds) phase that can be monitored directly.The refolding kinetics of the W7F, W7F/M131C, and W7F/L135C mutantswere monitored using stopped-flow fluorescence and CD measurements.The changes in W14 fluorescence emission during kinetic refoldingare shown in Figure 6. The kinetic traces forW7F and W7F/M131C are biphasic, reporting on the rates of the Ia → Ib and Ib → N foldingprocesses,23 and were fit to double-exponentialdecays. For W7F/L135C, the change in fluorescence emission duringthe slow phase (Ib → N) is of extremely small amplitude,because there is little change in fluorescence intensity between themolten globule and native state (Figure 5),and the fast phase dominates the stopped-flow trace (Figure 6). The fitted kinetic parameters are summarizedin Table 2.

Bottom Line: Fluorescence spectra of mutant proteins in which cysteine residues were introduced at several positions in the G and H helices show differential quenching of W14 fluorescence, providing direct evidence of translocation of the H helix relative to helices A and G in both the kinetic and equilibrium intermediates.Formation of an S108C-L135C disulfide prevents H helix translocation in the equilibrium molten globule by locking the G and H helices into their native register.By enforcing nativelike packing of the A, G, and H helices, the disulfide resolves local energetic frustration and facilitates transient docking of the E helix region onto the hydrophobic core but has only a small effect on the refolding rate.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Skaggs Institute of Chemical Biology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

ABSTRACT
Apomyoglobin folds via sequential helical intermediates that are formed by rapid collapse of the A, B, G, and H helix regions. An equilibrium molten globule with a similar structure is formed near pH 4. Previous studies suggested that the folding intermediates are kinetically trapped states in which folding is impeded by non-native packing of the G and H helices. Fluorescence spectra of mutant proteins in which cysteine residues were introduced at several positions in the G and H helices show differential quenching of W14 fluorescence, providing direct evidence of translocation of the H helix relative to helices A and G in both the kinetic and equilibrium intermediates. Förster resonance energy transfer measurements show that a 5-({2-[(acetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid acceptor coupled to K140C (helix H) is closer to Trp14 (helix A) in the equilibrium molten globule than in the native state, by a distance that is consistent with sliding of the H helix in an N-terminal direction by approximately one helical turn. Formation of an S108C-L135C disulfide prevents H helix translocation in the equilibrium molten globule by locking the G and H helices into their native register. By enforcing nativelike packing of the A, G, and H helices, the disulfide resolves local energetic frustration and facilitates transient docking of the E helix region onto the hydrophobic core but has only a small effect on the refolding rate. The apomyoglobin folding landscape is highly rugged, with several energetic bottlenecks that frustrate folding; relief of any one of the major identified bottlenecks is insufficient to speed progression to the transition state.

Show MeSH
Related in: MedlinePlus