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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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pH dependence of the quenching of thefluorescence of W14 inducedby the presence of cysteine at positions 111, 131, and 135. All proteinscontained the W7F mutation: W7F (filled black circles, black line),W7F/M131C (filled blue circles, blue line), W7F/L135C (filled orangetriangles, orange line), and W7F/I111C (filled magenta squares, magentaline). The maximal intensity of the fluorescence emission was recordedwith an excitation wavelength of 288 nm.
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fig5: pH dependence of the quenching of thefluorescence of W14 inducedby the presence of cysteine at positions 111, 131, and 135. All proteinscontained the W7F mutation: W7F (filled black circles, black line),W7F/M131C (filled blue circles, blue line), W7F/L135C (filled orangetriangles, orange line), and W7F/I111C (filled magenta squares, magentaline). The maximal intensity of the fluorescence emission was recordedwith an excitation wavelength of 288 nm.

Mentions: The pH dependence of the W14 fluorescence emission intensityin the W7F, W7F/I111C, W7F/M131C, and W7F/L135C mutants is shown inFigure 5. The emission maximum for W7F, correspondingto the pH at which the population of the molten globule intermediateis maximal, occurs at higher pH (4.4) than for wild-type apomyoglobin(pH 4.0) (Figure 4B), reflecting destabilizationof both the native state and the intermediate by the W7F mutation.49 The pH-induced unfolding curve for W7F/I111Cis very similar to that of W7F except for a slight increase in thelevel of fluorescence quenching in the pH 4.4 intermediate, suggestinga slight decrease in the distance between these side chains in theintermediate relative to the native state. By contrast, the titrationcurve of W7F/M131C is significantly different from that of W7F. AtpH 6, the intensity is much lower than that of W7F, which we attributeto an increased level of W14 fluorescence quenching because of theproximity of the W14 and C131 side chains in the native structure(the Met131 side chain makes direct contact with the indole ring inthe X-ray structure of holomyoglobin). However, the W14 fluorescenceintensity for W7F/M131C in the pH ∼4.5 intermediate is thesame as that for W7F, indicating no quenching by the Cys at position131 in the molten globule state. For W7F/L135C, the cysteine at 135is distant from the indole ring of W14 in the native state (∼7Å in the X-ray structure of holomyoglobin) and fails to quenchthe W14 fluorescence at pH 6. However, C135 strongly quenches thetryptophan fluorescence in the intermediate formed at pH ∼4.5(Figure 5), indicating close, non-native contactbetween the cysteine and tryptophan side chains in the equilibriummolten globule state. Taken together, the fluorescence quenching datafor this set of mutants provide strong support for a structural modelin which the H helix is displaced in an N-terminal direction in theequilibrium molten globule, bringing the side chain of residue 135into non-native contact with W14.


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

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

pH dependence of the quenching of thefluorescence of W14 inducedby the presence of cysteine at positions 111, 131, and 135. All proteinscontained the W7F mutation: W7F (filled black circles, black line),W7F/M131C (filled blue circles, blue line), W7F/L135C (filled orangetriangles, orange line), and W7F/I111C (filled magenta squares, magentaline). The maximal intensity of the fluorescence emission was recordedwith an excitation wavelength of 288 nm.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: pH dependence of the quenching of thefluorescence of W14 inducedby the presence of cysteine at positions 111, 131, and 135. All proteinscontained the W7F mutation: W7F (filled black circles, black line),W7F/M131C (filled blue circles, blue line), W7F/L135C (filled orangetriangles, orange line), and W7F/I111C (filled magenta squares, magentaline). The maximal intensity of the fluorescence emission was recordedwith an excitation wavelength of 288 nm.
Mentions: The pH dependence of the W14 fluorescence emission intensityin the W7F, W7F/I111C, W7F/M131C, and W7F/L135C mutants is shown inFigure 5. The emission maximum for W7F, correspondingto the pH at which the population of the molten globule intermediateis maximal, occurs at higher pH (4.4) than for wild-type apomyoglobin(pH 4.0) (Figure 4B), reflecting destabilizationof both the native state and the intermediate by the W7F mutation.49 The pH-induced unfolding curve for W7F/I111Cis very similar to that of W7F except for a slight increase in thelevel of fluorescence quenching in the pH 4.4 intermediate, suggestinga slight decrease in the distance between these side chains in theintermediate relative to the native state. By contrast, the titrationcurve of W7F/M131C is significantly different from that of W7F. AtpH 6, the intensity is much lower than that of W7F, which we attributeto an increased level of W14 fluorescence quenching because of theproximity of the W14 and C131 side chains in the native structure(the Met131 side chain makes direct contact with the indole ring inthe X-ray structure of holomyoglobin). However, the W14 fluorescenceintensity for W7F/M131C in the pH ∼4.5 intermediate is thesame as that for W7F, indicating no quenching by the Cys at position131 in the molten globule state. For W7F/L135C, the cysteine at 135is distant from the indole ring of W14 in the native state (∼7Å in the X-ray structure of holomyoglobin) and fails to quenchthe W14 fluorescence at pH 6. However, C135 strongly quenches thetryptophan fluorescence in the intermediate formed at pH ∼4.5(Figure 5), indicating close, non-native contactbetween the cysteine and tryptophan side chains in the equilibriummolten globule state. Taken together, the fluorescence quenching datafor this set of mutants provide strong support for a structural modelin which the H helix is displaced in an N-terminal direction in theequilibrium molten globule, bringing the side chain of residue 135into non-native contact with W14.

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