Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jun 17;53(23):3767-80.
doi: 10.1021/bi500478m. Epub 2014 Jun 4.

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

Phillip C Aoto  1 , Chiaki Nishimura, H Jane Dyson, Peter E Wright
Affiliations

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

Phillip C Aoto et al. Biochemistry. .

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Backbone chain trace of holomyoglobin. (A) Location of the residues, S108 and L135 (spheres), mutated to form the disulfide mutant. The S108–L135 Cα–Cα distance is shown. (B) Sites of the mutations used for fluorescence quenching and FRET studies and location of the two tryptophan residues. W7 (gray) was mutated to Phe for all of the FRET and fluorescence quenching experiments. The Cα positions of residues mutated in the FRET experiments are colored pink. The Cα positions of residues mutated in the cysteine fluorescence quenching experiments are colored yellow.
Figure 2
Figure 2
Comparison of chemical shifts of wild-type apomyoglobin and the oxidized S108C/L135C mutant. (A) Difference in the weighted average backbone HN and 15N chemical shifts {⟨Δδ⟩ = 1/2[(ΔδHN)2 + (ΔδN/5)2]1/2} between the wild type and oxidized two-cysteine mutant at pH 6.1. The location of the mutations is indicated by arrows. Horizontal lines represent the mean and (mean + 1 standard deviation) of the ⟨Δδ⟩ values. The inset shows major chemical shift changes at pH 6.1 mapped onto the structure of Mb. Positions of helices A–H are indicated by colored bars. Spheres colored according to the helix location represent the backbone N atoms of residues where the composite Δδ > (mean + 1 standard deviation). Yellow spheres show the positions of the mutated residues. (B) Difference in the weighted average backbone HN and 15N chemical shifts (⟨Δδ⟩) between the wild type and oxidized two-cysteine mutant at pH 4.1. The location of the mutations is indicated by arrows. Horizontal lines represent the mean and (mean + 1 standard deviation) of the ⟨Δδ⟩ values. The inset shows major chemical shift changes at pH 4.1 mapped onto the structure of Mb. Colored spheres represent the backbone N atoms of residues where the composite Δδ > (mean + 1 standard deviation).
Figure 3
Figure 3
(A) Secondary 13Cα chemical shifts (observed chemical shift minus the sequence-corrected random coil shift) for the wild type (black) and oxidized two-cysteine mutant (red) at pH 4.1. (B) Difference [ΔδCα = δCα(mutant) – δCα(WT)] between the 13Cα chemical shifts in panel A. (C) Heteronuclear {1H}–15N NOE at pH 4.1 for the wild type (black) and oxidized two-cysteine mutant (red).
Figure 4
Figure 4
Variation in CD and fluorescence signals with pH and urea. (A) pH dependence of the mean residue ellipticity at 222 nm for wild-type apomyoglobin (black) and the oxidized two-cysteine mutant (red). Solid curves are fit to the data using the method of least squares to a three-state unfolding model. (B) pH dependence of the fluorescence signal at the emission maximum upon excitation at 288 nm for wild-type apomyoglobin (black) and the oxidized two-cysteine mutant (red). Solid curves are fit to the data using the method of damped least squares to a three-state unfolding model. (C) Urea dependence of the mean residue ellipticity at 222 nm for wild-type apomyoglobin (black) at pH 6.1 (filled squares) and pH 4.1 (empty squares), the oxidized two-cysteine mutant (red) at pH 6.1 (filled circles) and pH 4.1 (empty circles), and the reduced two-cysteine mutant (blue) at pH 6.1 (filled triangles). Data were fit by the method of least squares to a two-state unfolding model.
Figure 5
Figure 5
pH dependence of the quenching of the fluorescence of W14 induced by the presence of cysteine at positions 111, 131, and 135. All proteins contained the W7F mutation: W7F (filled black circles, black line), W7F/M131C (filled blue circles, blue line), W7F/L135C (filled orange triangles, orange line), and W7F/I111C (filled magenta squares, magenta line). The maximal intensity of the fluorescence emission was recorded with an excitation wavelength of 288 nm.
Figure 6
Figure 6
Fluorescence decay after a pH jump from pH 2.2 to 6.0 for W7F (black), W7F/M131C (blue), and W7F/L135C (orange). The stopped-flow traces show total fluorescence with a 320 nm cutoff filter. The excitation wavelength was 288 nm.
Figure 7
Figure 7
Stopped-flow fluorescence data. (A) Intensity decay curves for wild-type apomyoglobin (black), the oxidized two-cysteine mutant (red), and the reduced two-cysteine mutant (blue) from a urea jump experiment (8 M → 1.3 M). Curves have been scaled to give a similar final value of fluorescence intensity, to allow visual comparison of the relative rates. Estimates of burst phase amplitudes cannot be obtained from these data. (B) Fluorescence decay for wild-type apomyoglobin and the oxidized two-cysteine mutant following a pH jump (from pH 2.5 to 6.0).
Figure 8
Figure 8
Direct emission from the incorporated IAEDANS as a function of pH for the mutants indicated. A set of mutants that showed no additional AEDANS fluorescence increase between pH 4 and 6 are bracketed. The excitation wavelength was set at 338 nm, and the fluorescence emission was observed at the emission maximum close to 480 nm.
Figure 9
Figure 9
Fluorescence spectra of the AEDANS-substituted mutants under various conditions of pH and presence of denaturant: (A) K77C-A and (B) K140C-A.
Figure 10
Figure 10
(A) pH dependence of FRET from W14 (A helix) to the AEDANS fluorescence acceptor covalently attached at the mutated cysteine residue for mutants K102C-A (red), E105C-A (green), F106C-A (black), R139C-A (orange), and K140C-A (blue). The FRET efficiency is given by E = 1 – F/F0, where F and F0 are the W14 emission intensities, measured at the fluorescence maximum close to 338 nm, in the presence and absence of the coupled AEDANS acceptor, respectively. (B) Distances derived from fluorescence energy transfer. The excitation wavelength was 280 nm. Distances were calculated from the FRET efficiencies in panel A using the Förster equation with an R0 of 22 Å and a κ2 of 0.67.
Figure 11
Figure 11
Schematic diagram illustrating how a translocation of the H helix toward the N-terminus by approximately one helical turn decreases the distance between a fluorescence acceptor at position 140 and the donor W14 in the molten globule intermediate compared to the folded state. The backbone structure of myoglobin is colored to show the regions of the protein that are folded and protect amides from exchange in the burst phase intermediate.,, Helix colors correspond to those in Figure 1. The distances between the Cα atom of residue 140 in the “native” and “translocated” forms of the H helix are shown schematically as green and yellow lines, respectively. The position of K140 in the translocated H helix is modeled by the Cα atom of L137 for illustrative purposes.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Udgaonkar J. B.; Baldwin R. L. (1988) NMR evidence for an early framework intermediate on the folding pathway of ribonuclease A. Nature 335, 694–699. - PubMed
    1. Roder H.; Elöve G. A.; Englander S. W. (1988) Structural characterization of folding intermediates in cytochrome c by H-exchange labelling and proton NMR. Nature 335, 700–704. - PMC - PubMed
    1. Lyon C. E.; Suh E. S.; Dobson C. M.; Hore P. J. (2002) Probing the exposure of tyrosine and tryptophan residues in partially folded proteins and folding intermediates by CIDNP pulse-labeling. J. Am. Chem. Soc. 124, 13018–13024. - PubMed
    1. Balbach J.; Forge V.; van Nuland N. A. J.; Winder S. L.; Hore P. J.; Dobson C. M. (1995) Following protein folding in real time using NMR spectroscopy. Nat. Struct. Biol. 2, 865–870. - PubMed
    1. Balbach J. (2000) Compaction during protein folding studied by real-time NMR diffusion experiments. J. Am. Chem. Soc. 122, 5887–5888.

Publication types

MeSH terms

LinkOut - more resources