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Collapse of a long axis: single-molecule Förster resonance energy transfer and serpin equilibrium unfolding.

Liu L, Werner M, Gershenson A - Biochemistry (2014)

Bottom Line: Most of the structural remodeling required for function occurs along the long axis, while expansion of the short axes is associated with misfolded, inactive forms.Our spFRET studies agree with other equilibrium unfolding studies that found that the region around one of the β strands, s5A, which helps define the long axis and must move for functionally required loop insertion, unfolds at low denaturant concentrations.This supports a connection between functionally important structural lability and unfolding in the inhibitory serpins.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Brandeis University , Waltham, Massachusetts 02453, United States.

ABSTRACT
The energy required for mechanical inhibition of target proteases is stored in the native structure of inhibitory serpins and accessed by serpin structural remodeling. The overall serpin fold is ellipsoidal with one long and two short axes. Most of the structural remodeling required for function occurs along the long axis, while expansion of the short axes is associated with misfolded, inactive forms. This suggests that ellipticity, as typified by the long axis, may be important for both function and folding. Placement of donor and acceptor fluorophores approximately along the long axis or one of the short axes allows single-pair Förster resonance energy transfer (spFRET) to report on both unfolding transitions and the time-averaged shape of different conformations. Equilibrium unfolding and refolding studies of the well-characterized inhibitory serpin α1-antitrypsin reveal that the long axis collapses in the folding intermediates while the monitored short axis expands. These energetically distinct intermediates are thus more spherical than the native state. Our spFRET studies agree with other equilibrium unfolding studies that found that the region around one of the β strands, s5A, which helps define the long axis and must move for functionally required loop insertion, unfolds at low denaturant concentrations. This supports a connection between functionally important structural lability and unfolding in the inhibitory serpins.

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Related in: MedlinePlus

α1AT unfolding is different along the short andlong axes. spFRET histograms for the unfolding of 2Cys47 (A) and 2Cys313(B) in increasing concentrations of GdmCl as indicated on the plots.The total Gaussian fits to the histograms are indicated by red lines,and the resulting single Gaussians for the native, intermediate, unfolded,and zero peaks are indicated by green, blue, black, and yellow lines,respectively. The distance between residues 232 and 47 (2Cys47) increasesas α1AT unfolds, while the distance between residues232 and 313 (2Cys313) first decreases and then increases. (C and D)Centers, ⟨Em⟩, and half-widths,σm, of the spFRET peaks, determined from the Gaussianfits, as a function of GdmCl concentration for 2Cys47 (C) and 2Cys313(D). Data for the native, intermediate, and unfolded species are coloredgreen, blue, and black, respectively. Filled symbols were determinedfrom unfolding experiments, while empty symbols were determined fromrefolding experiments.
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fig3: α1AT unfolding is different along the short andlong axes. spFRET histograms for the unfolding of 2Cys47 (A) and 2Cys313(B) in increasing concentrations of GdmCl as indicated on the plots.The total Gaussian fits to the histograms are indicated by red lines,and the resulting single Gaussians for the native, intermediate, unfolded,and zero peaks are indicated by green, blue, black, and yellow lines,respectively. The distance between residues 232 and 47 (2Cys47) increasesas α1AT unfolds, while the distance between residues232 and 313 (2Cys313) first decreases and then increases. (C and D)Centers, ⟨Em⟩, and half-widths,σm, of the spFRET peaks, determined from the Gaussianfits, as a function of GdmCl concentration for 2Cys47 (C) and 2Cys313(D). Data for the native, intermediate, and unfolded species are coloredgreen, blue, and black, respectively. Filled symbols were determinedfrom unfolding experiments, while empty symbols were determined fromrefolding experiments.

Mentions: In0 M GdmCl, spFREThistograms for doubly labeled 2Cys47 show two peaks, a small, “zero”peak arising from proteins containing only AF488 (donor) and proteinsin which TR (acceptor) has photobleached as well as a much largerpeak with an apparent mean efficiency, ⟨Em⟩, of 0.95 (0.96 when corrected for γ) (Figure 3A). The short distance, ∼27 Å, betweenresidue 47 and Cys232 in the native structure would yield a nativestate spFRET signal centered at 0.97. However, the five-carbon (AF488)or two-carbon (TR) flexible chains linking the fluorophores to theCys residues can increase this distance, and the peak centered at0.95 efficiency with a half-width of 0.04 can easily be assigned tothe α1AT native state (Figure 3A).


Collapse of a long axis: single-molecule Förster resonance energy transfer and serpin equilibrium unfolding.

Liu L, Werner M, Gershenson A - Biochemistry (2014)

α1AT unfolding is different along the short andlong axes. spFRET histograms for the unfolding of 2Cys47 (A) and 2Cys313(B) in increasing concentrations of GdmCl as indicated on the plots.The total Gaussian fits to the histograms are indicated by red lines,and the resulting single Gaussians for the native, intermediate, unfolded,and zero peaks are indicated by green, blue, black, and yellow lines,respectively. The distance between residues 232 and 47 (2Cys47) increasesas α1AT unfolds, while the distance between residues232 and 313 (2Cys313) first decreases and then increases. (C and D)Centers, ⟨Em⟩, and half-widths,σm, of the spFRET peaks, determined from the Gaussianfits, as a function of GdmCl concentration for 2Cys47 (C) and 2Cys313(D). Data for the native, intermediate, and unfolded species are coloredgreen, blue, and black, respectively. Filled symbols were determinedfrom unfolding experiments, while empty symbols were determined fromrefolding experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4020580&req=5

fig3: α1AT unfolding is different along the short andlong axes. spFRET histograms for the unfolding of 2Cys47 (A) and 2Cys313(B) in increasing concentrations of GdmCl as indicated on the plots.The total Gaussian fits to the histograms are indicated by red lines,and the resulting single Gaussians for the native, intermediate, unfolded,and zero peaks are indicated by green, blue, black, and yellow lines,respectively. The distance between residues 232 and 47 (2Cys47) increasesas α1AT unfolds, while the distance between residues232 and 313 (2Cys313) first decreases and then increases. (C and D)Centers, ⟨Em⟩, and half-widths,σm, of the spFRET peaks, determined from the Gaussianfits, as a function of GdmCl concentration for 2Cys47 (C) and 2Cys313(D). Data for the native, intermediate, and unfolded species are coloredgreen, blue, and black, respectively. Filled symbols were determinedfrom unfolding experiments, while empty symbols were determined fromrefolding experiments.
Mentions: In0 M GdmCl, spFREThistograms for doubly labeled 2Cys47 show two peaks, a small, “zero”peak arising from proteins containing only AF488 (donor) and proteinsin which TR (acceptor) has photobleached as well as a much largerpeak with an apparent mean efficiency, ⟨Em⟩, of 0.95 (0.96 when corrected for γ) (Figure 3A). The short distance, ∼27 Å, betweenresidue 47 and Cys232 in the native structure would yield a nativestate spFRET signal centered at 0.97. However, the five-carbon (AF488)or two-carbon (TR) flexible chains linking the fluorophores to theCys residues can increase this distance, and the peak centered at0.95 efficiency with a half-width of 0.04 can easily be assigned tothe α1AT native state (Figure 3A).

Bottom Line: Most of the structural remodeling required for function occurs along the long axis, while expansion of the short axes is associated with misfolded, inactive forms.Our spFRET studies agree with other equilibrium unfolding studies that found that the region around one of the β strands, s5A, which helps define the long axis and must move for functionally required loop insertion, unfolds at low denaturant concentrations.This supports a connection between functionally important structural lability and unfolding in the inhibitory serpins.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Brandeis University , Waltham, Massachusetts 02453, United States.

ABSTRACT
The energy required for mechanical inhibition of target proteases is stored in the native structure of inhibitory serpins and accessed by serpin structural remodeling. The overall serpin fold is ellipsoidal with one long and two short axes. Most of the structural remodeling required for function occurs along the long axis, while expansion of the short axes is associated with misfolded, inactive forms. This suggests that ellipticity, as typified by the long axis, may be important for both function and folding. Placement of donor and acceptor fluorophores approximately along the long axis or one of the short axes allows single-pair Förster resonance energy transfer (spFRET) to report on both unfolding transitions and the time-averaged shape of different conformations. Equilibrium unfolding and refolding studies of the well-characterized inhibitory serpin α1-antitrypsin reveal that the long axis collapses in the folding intermediates while the monitored short axis expands. These energetically distinct intermediates are thus more spherical than the native state. Our spFRET studies agree with other equilibrium unfolding studies that found that the region around one of the β strands, s5A, which helps define the long axis and must move for functionally required loop insertion, unfolds at low denaturant concentrations. This supports a connection between functionally important structural lability and unfolding in the inhibitory serpins.

Show MeSH
Related in: MedlinePlus