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

Biophys J. 2018 Jul 3;115(1):46-58. doi: 10.1016/j.bpj.2018.05.028.


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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Biomechanical Phenomena
  • Enzyme Stability
  • Escherichia coli / enzymology
  • Mechanical Phenomena*
  • Molecular Dynamics Simulation
  • Phosphoglycerate Kinase / chemistry*
  • Phosphoglycerate Kinase / metabolism*
  • Protein Domains
  • Protein Unfolding*
  • Saccharomyces cerevisiae / enzymology


  • Phosphoglycerate Kinase