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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 spectra of the AEDANS-substitutedmutants under variousconditions of pH and presence of denaturant: (A) K77C-A and (B) K140C-A.
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fig9: Fluorescence spectra of the AEDANS-substitutedmutants under variousconditions of pH and presence of denaturant: (A) K77C-A and (B) K140C-A.

Mentions: Fluorescenceemission spectra of several of the AEDANS-derivatized W7F mutantswere recorded in 6 M urea, in the acid-denatured state at pH 2, andin the molten globule and native states at pH 4 and 6, respectively.Representative spectra for the AEDANS-coupled K77C-A and K140C-A mutantsare shown in Figure 9. The fluorescence spectrain 6 M urea (black trace) show two maxima, corresponding to fluorescenceemission by W14 (at ∼350 nm) and AEDANS (at ∼490 nm).Even when the protein is unfolded in 6 M urea, a basal level of resonanceenergy transfer occurs from W14 to AEDANS. This energy transfer isrelatively independent of the location of the AEDANS acceptor andarises through transient sampling of partially collapsed states withinthe unfolded conformational ensemble. Energy transfer also occursin the acid-unfolded state at pH 2 (red traces), but its efficiencyis strongly dependent upon the location of the AEDANS probe. For K77C-A,there is very little difference between the spectra in 6 M urea andin the absence of urea at pH 2, but at pH 4 (blue trace) and pH 6(green trace), the emission at ∼480 nm is greatly increasedand that at ∼330 nm decreased, because of efficient energytransfer in the collapsed molten globule and native states. By contrast,the spectrum of K140C-A at pH 2 in the absence of urea is markedlydifferent from that of the urea-denatured form, with a greater emissionat ∼480 nm due to FRET arising from transiently collapsed statesin which there are contacts between the A and H helix regions. Theseinteractions have been described previously on the basis of paramagneticbroadening effects of nitroxide spin-label probes.52 The 480 nm emission at pH 4 is greater than that at pH6, showing that the W14 donor and AEDANS acceptor are closer in themolten globule state than in the native state. These observationsprovide important clues about the structure of the intermediate aswell as the presence of long-range contacts involving W14 even inthe pH 2 unfolded state.


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

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

Fluorescence spectra of the AEDANS-substitutedmutants under variousconditions of pH and presence of denaturant: (A) K77C-A and (B) K140C-A.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Fluorescence spectra of the AEDANS-substitutedmutants under variousconditions of pH and presence of denaturant: (A) K77C-A and (B) K140C-A.
Mentions: Fluorescenceemission spectra of several of the AEDANS-derivatized W7F mutantswere recorded in 6 M urea, in the acid-denatured state at pH 2, andin the molten globule and native states at pH 4 and 6, respectively.Representative spectra for the AEDANS-coupled K77C-A and K140C-A mutantsare shown in Figure 9. The fluorescence spectrain 6 M urea (black trace) show two maxima, corresponding to fluorescenceemission by W14 (at ∼350 nm) and AEDANS (at ∼490 nm).Even when the protein is unfolded in 6 M urea, a basal level of resonanceenergy transfer occurs from W14 to AEDANS. This energy transfer isrelatively independent of the location of the AEDANS acceptor andarises through transient sampling of partially collapsed states withinthe unfolded conformational ensemble. Energy transfer also occursin the acid-unfolded state at pH 2 (red traces), but its efficiencyis strongly dependent upon the location of the AEDANS probe. For K77C-A,there is very little difference between the spectra in 6 M urea andin the absence of urea at pH 2, but at pH 4 (blue trace) and pH 6(green trace), the emission at ∼480 nm is greatly increasedand that at ∼330 nm decreased, because of efficient energytransfer in the collapsed molten globule and native states. By contrast,the spectrum of K140C-A at pH 2 in the absence of urea is markedlydifferent from that of the urea-denatured form, with a greater emissionat ∼480 nm due to FRET arising from transiently collapsed statesin which there are contacts between the A and H helix regions. Theseinteractions have been described previously on the basis of paramagneticbroadening effects of nitroxide spin-label probes.52 The 480 nm emission at pH 4 is greater than that at pH6, showing that the W14 donor and AEDANS acceptor are closer in themolten globule state than in the native state. These observationsprovide important clues about the structure of the intermediate aswell as the presence of long-range contacts involving W14 even inthe pH 2 unfolded state.

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