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.

Mapping Multiple Distances in a Multidomain Protein for the Identification of Folding Intermediates

“…In the past, chemical-as well as heat-and cold-induced unfolding of yPGK has been studied extensively using traditional bulk methods, including circular dichroism (CD), tryptophan fluorescence, stopped-flow fluorescence, and FRET (22)(23)(24)(25)(26). More recently, these studies were also extended by single-molecule measurement (13,27,28). To obtain a more detailed picture about the conformational changes of yPGK, we pursued our previous smFRET studies.…”

“…The use of smFRET offers a unique tool to map conformational changes within the protein structure, thus allowing one to observe the distribution in populations. Although most of these studies dealt with small single-domain proteins, more recently, studies with multidomain proteins have attracted increasing interest ( 8 , 9 , 10 , 11 , 12 , 13 ). Multidomain proteins exhibit, typically, a much higher complexity of the folding landscape than that of single-domain proteins.…”

Combination of Click Chemistry and Enzymatic Ligation for Stable and Efficient Protein Immobilization for Single-Molecule Force Spectroscopy

“…To confirm the folding and stability of the immobilized eGFP, we performed the AFM-SMFS unfolding experiment. AFM manipulates a single molecule [27][28][29][30][31] or complexes mechanically, [32][33][34] which can (un)fold the protein, [35][36][37][38] break molecular interactions, [39][40][41] and chemically bond. [42][43][44][45][46][47][48] In our previous design, cohesin (Coh) was fused to the eGFP, which binds to the Xmod-dockerin (XDoc) site specifically and reversibly with a rupture force of ∼600 pN.…”

Protein Cross-Interactions for Efficient Photosynthesis in the Cassava Cultivar SC205 Relative to Its Wild Species

“…PGK is crucial for glycolysis and is a highly conserved enzyme in plants. 40 It links the TCA cycle and the pentose phosphate pathway, and interacts with RCA. In Figure 6, PGK interacts with AOR and FBA2 which are involved in the TCA cycle as well as with RPE that is associated with the pentose phosphate pathway.…”

“…In plants, ATP is produced through many pathways, such as the TCA cycle, glycolysis, and the pentose phosphate pathway. PGK is crucial for glycolysis and is a highly conserved enzyme in plants . It links the TCA cycle and the pentose phosphate pathway, and interacts with RCA.…”