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How force unfolding differs from chemical denaturation.

ABSTRACT: Single-molecule force spectroscopies are remarkable tools for studying protein folding and unfolding, but force unfolding explores protein configurations that are potentially very different from the ones traditionally explored in chemical or thermal denaturation. Understanding these differences is crucial because such configurations serve as starting points of folding studies, and thus can affect both the folding mechanism and the kinetics. Here we provide a detailed comparison of both chemically induced and force-induced unfolded state ensembles of ubiquitin based on extensive, all-atom simulations of the protein either extended by force or denatured by urea. As expected, the respective unfolded states are very different on a macromolecular scale, being fully extended under force with no contacts and partially extended in urea with many nonnative contacts. The amount of residual secondary structure also differs: A significant population of ?-helices is found in chemically denatured configurations but such helices are absent under force, except at the lowest applied force of 30 pN where short helices form transiently. We see that typical-size helices are unstable above this force, and ?-sheets cannot form. More surprisingly, we observe striking differences in the backbone dihedral angle distributions for the protein unfolded under force and the one unfolded by denaturant. A simple model based on the dialanine peptide is shown to not only provide an explanation for these striking differences but also illustrates how the force dependence of the protein dihedral angle distributions give rise to the worm-like chain behavior of the chain upon force.

SUBMITTER: Stirnemann G

PROVIDER: S-EPMC3948277 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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How force unfolding differs from chemical denaturation.

Stirnemann Guillaume G Kang Seung-gu SG Zhou Ruhong R Berne Bruce J BJ

Proceedings of the National Academy of Sciences of the United States of America 20140218 9

Single-molecule force spectroscopies are remarkable tools for studying protein folding and unfolding, but force unfolding explores protein configurations that are potentially very different from the ones traditionally explored in chemical or thermal denaturation. Understanding these differences is crucial because such configurations serve as starting points of folding studies, and thus can affect both the folding mechanism and the kinetics. Here we provide a detailed comparison of both chemicall ...[more]

PMID: 24550471

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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.

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How force unfolding differs from chemical denaturation.

Publications

How force unfolding differs from chemical denaturation.

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