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Equilibrium folding dynamics of meACP in water, heavy water, and low concentration of urea.

ABSTRACT: Many proteins fold in apparent two-state behavior, as partially folded intermediates only transiently accumulate and easily escape detection. Besides a native form and a mainly unfolded form, we captured a partially unfolded form of an acyl carrier protein from Micromonospora echinospora (meACP) in the folding/unfolding equilibrium using chemical exchange saturation transfer NMR experiments. The C-terminal region of the partially unfolded form is mainly folded and the N-terminal is unfolded. Furthermore, to understand how the folding process of meACP is influenced by solvent environments, we compared the folding dynamics of meACP in D2O, H2O and low concentration of urea. As the environment becomes more denaturing from D2O to H2O and then to urea, the unfolded state becomes increasingly populated, and the folding rate decreases. Adding a small amount of urea, which does not change solvent viscosity, has little effects on the unfolding rates, while changing H2O to D2O reduces the unfolding rates possibly due to the increase of solvent viscosity. The quantified solvent effects on the protein folding Gibbs energy and activation energy suggest that the transition state of folding may have a similar structure to the native state of the protein.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC5700953 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Equilibrium folding dynamics of meACP in water, heavy water, and low concentration of urea.

Zhou Yang Y Yang Daiwen D

Scientific reports 20171123 1

Many proteins fold in apparent two-state behavior, as partially folded intermediates only transiently accumulate and easily escape detection. Besides a native form and a mainly unfolded form, we captured a partially unfolded form of an acyl carrier protein from Micromonospora echinospora (meACP) in the folding/unfolding equilibrium using chemical exchange saturation transfer NMR experiments. The C-terminal region of the partially unfolded form is mainly folded and the N-terminal is unfolded. Fur ...[more]

PMID: 29170533

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Project description:Earliest events in the aggregation process, such as single molecule reconfiguration, are extremely important and the most difficult to characterize in experiments. To this end, we have used well-tempered bias exchange metadynamics simulations to determine the equilibrium ensembles of an insulin molecule under amyloidogenic conditions of low pH and high temperature. A bin-based clustering method that uses statistics accumulated in bias exchange metadynamics trajectories was employed to construct a detailed thermodynamic and kinetic model of insulin folding. The highest lifetime, lowest free-energy ensemble identified consisted of native conformations adopted by a folded insulin monomer in solution, namely, the R-, the Rf-, and the T-states of insulin. The lowest free-energy structure had a root mean square deviation of only 0.15 nm from native x-ray structure. The second longest-lived metastable state was an unfolded, compact monomer with little similarity to the native structure. We have identified three additional long-lived, metastable states from the bin-based model. We then carried out an exhaustive structural characterization of metastable states on the basis of tertiary contact maps and per-residue accessible surface areas. We have also determined the lowest free-energy path between two longest-lived metastable states and confirm earlier findings of non-two-state folding for insulin through a folding intermediate. The ensemble containing the monomeric intermediate retained 58% of native hydrophobic contacts, however, accompanied by a complete loss of native secondary structure. We have discussed the relative importance of nativelike versus nonnative tertiary contacts for the folding transition. We also provide a simple measure to determine the importance of an individual residue for folding transition. Finally, we have compared and contrasted this intermediate with experimental data obtained in spectroscopic, crystallographic, and calorimetric measurements during early stages of insulin aggregation. We have also determined stability of monomeric insulin by incubation at a very low concentration to isolate protein-protein interaction effects.

Dataset Information

Equilibrium folding dynamics of meACP in water, heavy water, and low concentration of urea.

Publications

Equilibrium folding dynamics of meACP in water, heavy water, and low concentration of urea.

Similar Datasets

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