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Updated: Jul 11, 2025
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Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes
Published on: January 16, 2016
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Circular permutation at azurin's active site slows down its folding.
Debanjana Das1, Sri Rama Koti Ainavarapu2
1Department of Chemical Sciences, Tata Institute of Fundamental Research, Dr. Homi Bhabha Road, Colaba, Mumbai, 400005, India.
Summary
Circular permutation (CP) alters protein termini, affecting stability and folding rates. This study on metalloprotein azurin shows CP destabilizes the protein and slows folding, highlighting sequence and termini importance.
Area of Science:
- Protein engineering and biophysics
- Metalloprotein structure and function
- Protein folding dynamics
Background:
- Circular permutation (CP) rearranges protein sequences, creating new termini while often preserving overall structure.
- CP can impact protein stability and folding kinetics, with limited studies on metalloproteins.
- Azurin, a bacterial metalloprotein, serves as a model to investigate CP effects in metal-containing proteins.
Purpose of the Study:
- To investigate the effects of circular permutation within the metal-binding site of azurin (cpF114) on its stability, folding, and unfolding rates.
- To compare the folding pathways of apo- and Zn2+-bound CP azurin with wild-type (WT) azurin.
- To explore CP as a tool for modifying protein energy landscapes and folding pathways.
Main Methods:
- Circular permutation of bacterial azurin within the metal-binding site (cpF114).
- Spectroscopic analysis (fluorescence and circular dichroism) to study protein stability and folding/unfolding kinetics.
- Comparison of CP azurin with WT azurin in both apo- and metal-bound states.
Main Results:
- Circular permutation introduced destabilizing effects on azurin.
- Zn2+-bound CP azurin exhibited significantly slower folding rates compared to Zn2+-WT azurin and apo-proteins.
- cpF114 displayed apparent two-state equilibrium unfolding but possessed an off-pathway kinetic intermediate, unlike cpN42 which showed equilibrium and kinetic intermediates.
Conclusions:
- Circular permutation can alter protein folding pathways and modify the energy landscape.
- Protein termini positions and sequence are critical determinants of metalloprotein stability and folding dynamics.
- WT azurin's faster folding may reflect evolutionary optimization for metal-bound states compared to its permuted forms.
Keywords:
Chemical denaturationChevron plotsOff-pathway folding intermediateProtein foldingTime-resolved fluorescenceZinc-bindingMore Related Videos
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