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Multiple Unfolding Intermediates Obtained by Molecular Dynamic Simulations under Stretching for Immunoglobulin-Binding Domain of Protein G.

ABSTRACT: We have studied the mechanical properties of the immunoglobulin-binding domain of protein G at the atomic level under stretching at constant velocity using molecular dynamics simulations. We have found that the unfolding process can occur either in a single step or through intermediate states. Analysis of the trajectories from the molecular dynamic simulations showed that the mechanical unfolding of the immunoglobulin-binding domain of protein G is triggered by the separation of the terminal beta-strands and the order in which the secondary-structure elements break is practically the same in two- and multi-state events and at the different extension velocities studied. It is seen from our analysis of 24 trajectories that the theoretical pathway of mechanical unfolding for the immunoglobulin-binding domain of protein G does not coincide with that proposed in denaturant studies in the absence of force.

SUBMITTER: Glyakina AV

PROVIDER: S-EPMC2793399 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Multiple Unfolding Intermediates Obtained by Molecular Dynamic Simulations under Stretching for Immunoglobulin-Binding Domain of Protein G.

Glyakina Anna V AV Balabaev Nikolai K NK Galzitskaya Oxana V OV

The open biochemistry journal 20091123

We have studied the mechanical properties of the immunoglobulin-binding domain of protein G at the atomic level under stretching at constant velocity using molecular dynamics simulations. We have found that the unfolding process can occur either in a single step or through intermediate states. Analysis of the trajectories from the molecular dynamic simulations showed that the mechanical unfolding of the immunoglobulin-binding domain of protein G is triggered by the separation of the terminal bet ...[more]

PMID: 20037652

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Project description:Alkaline phosphatase is an enzyme with a typical alpha/beta hydrolase fold. The conformational stability of the human placental alkaline phosphatase was examined with the chemical denaturant urea. The red shifts of fluorescence spectra show a complex unfolding process involving multiple equilibrium intermediates indicating differential stability of the subdomains of the enzyme. None of these unfolding intermediates were observed in the presence of 83 mM NaCl, indicating the importance of ionic interactions in the stabilization of the unfolding intermediates. Guanidinium chloride, on the other hand, could stabilize one of the unfolding intermediates, which is not a salt effect. Some of the unfolding intermediates were also observed in circular dichroism spectroscopy, which clearly indicates steady loss of helical structure during unfolding, but very little change was observed for the beta strand content until the late stage of the unfolding process. The enzyme does not lose its phosphate-binding ability after substantial tertiary structure changes, suggesting that the substrate-binding region is more resistant to chemical denaturant than the other structural domains. Global analysis of the fluorescence spectral change demonstrated the following folding-unfolding process of the enzyme: N <--> I(1) <--> I(2) <--> I(3) <--> I(4) <--> I(5) <--> D. These discrete intermediates are stable at urea concentrations of 2.6, 4.1, 4.7, 5.5, 6.6, and 7.7 M, respectively. These intermediates are further characterized by acrylamide and/or potassium iodide quenching of the intrinsic fluorescence of the enzyme and by the hydrophobic probes, 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The stepwise unfolding process was interpreted by the folding energy landscape in terms of the unique structure of the enzyme. The rigid central beta-strand domain is surrounded by the peripheral alpha-helical and coil structures, which are marginally stable toward a chemical denaturant.

Project description:Understanding the relationships between conformations of proteins and their stabilities is one key to address the protein folding paradigm. The free energy change (ΔG) of unfolding reactions of proteins is measured by traditional denaturation methods and native hydrogen-deuterium (H/D) exchange methods. However, the free energy of unfolding (ΔG(U)) and the free energy of exchange (ΔG(HX)) of proteins are not in good agreement, though the experimental conditions of both methods are well matching to each other. The anomaly is due to any one or combinations of the following reasons: (i) effects of cis-trans proline isomerisation under equilibrium unfolding reactions of proteins (ii) inappropriateness in accounting the baselines of melting curves (iii) presence of cryptic intermediates, which may elude the melting curve analysis and (iv) existence of higher energy metastable states in the H/D exchange reactions of proteins. Herein, we have developed a novel computational tool, OneG, which accounts the discrepancy between ΔG(U) and ΔG(HX) of proteins by systematically accounting all the four factors mentioned above. The program is fully automated and requires four inputs: three-dimensional structures of proteins, ΔG(U), ΔG(U)(*) and residue-specific ΔG(HX) determined under EX2-exchange conditions in the absence of denaturants. The robustness of the program has been validated using experimental data available for proteins such as cytochrome c and apocytochrome b(562) and the data analyses revealed that cryptic intermediates of the proteins detected by the experimental methods and the cryptic intermediates predicted by the OneG for those proteins were in good agreement. Furthermore, using OneG, we have shown possible existence of cryptic intermediates and metastable states in the unfolding pathways of cardiotoxin III and cobrotoxin, respectively, which are homologous proteins. The unique application of the program to map the unfolding pathways of proteins under native conditions have been brought into fore and the program is publicly available at http://sblab.sastra.edu/oneg.html.

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Multiple Unfolding Intermediates Obtained by Molecular Dynamic Simulations under Stretching for Immunoglobulin-Binding Domain of Protein G.

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Multiple Unfolding Intermediates Obtained by Molecular Dynamic Simulations under Stretching for Immunoglobulin-Binding Domain of Protein G.

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