Dataset Information

Unraveling the Mechanical Unfolding Pathways of a Multidomain Protein: Phosphoglycerate Kinase.

ABSTRACT: Phosphoglycerate kinase (PGK) is a highly conserved enzyme that is crucial for glycolysis. PGK is a monomeric protein composed of two similar domains and has been the focus of many studies for investigating interdomain interactions within the native state and during folding. Previous studies used traditional biophysical methods (such as circular dichroism, tryptophan fluorescence, and NMR) to measure signals over a large ensemble of molecules, which made it difficult to observe transient changes in stability or structure during unfolding and refolding of single molecules. Here, we unfold single molecules of PGK using atomic force spectroscopy and steered molecular dynamic computer simulations to examine the conformational dynamics of PGK during its unfolding process. Our results show that after the initial forced separation of its domains, yeast PGK (yPGK) does not follow a single mechanical unfolding pathway; instead, it stochastically follows two distinct pathways: unfolding from the N-terminal domain or unfolding from the C-terminal domain. The truncated yPGK N-terminal domain unfolds via a transient intermediate, whereas the structurally similar isolated C-terminal domain has no detectable intermediates throughout its mechanical unfolding process. The N-terminal domain in the full-length yPGK displays a strong unfolding intermediate 13% of the time, whereas the truncated domain (yPGKNT) transitions through the intermediate 81% of the time. This effect indicates that the mechanical properties of yPGK cannot be simply deduced from the mechanical properties of its constituents. We also find that Escherichia coli PGK is significantly less mechanically stable as compared to yPGK, contrary to bulk unfolding measurements. Our results support the growing body of observations that the folding behavior of multidomain proteins is difficult to predict based solely on the studies of isolated domains.

SUBMITTER: Li Q

PROVIDER: S-EPMC6035294 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Unraveling the Mechanical Unfolding Pathways of a Multidomain Protein: Phosphoglycerate Kinase.

Li Qing Q Scholl Zackary N ZN Marszalek Piotr E PE

Biophysical journal 20180701 1

Phosphoglycerate kinase (PGK) is a highly conserved enzyme that is crucial for glycolysis. PGK is a monomeric protein composed of two similar domains and has been the focus of many studies for investigating interdomain interactions within the native state and during folding. Previous studies used traditional biophysical methods (such as circular dichroism, tryptophan fluorescence, and NMR) to measure signals over a large ensemble of molecules, which made it difficult to observe transient changes ...[more]

PMID: 29972811

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Project description:Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from ∼30 to ∼150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties.

Dataset Information

Unraveling the Mechanical Unfolding Pathways of a Multidomain Protein: Phosphoglycerate Kinase.

Publications

Unraveling the Mechanical Unfolding Pathways of a Multidomain Protein: Phosphoglycerate Kinase.

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