Yeast Phosphoglycerate Kinase Research Articles

Metastable States in the Hinge-Bending Landscape of an Enzyme in an Atomistic Cytoplasm Simulation.

Many enzymes undergo major conformational changes to function in cells, particularly when they bind to more than one substrate. We quantify the large-amplitude hinge-bending landscape of human phosphoglycerate kinase (PGK) in a human cytoplasm. Approximately 70 μs of all-atom simulations, upon coarse graining, reveal three metastable states of PGK with different hinge angle distributions and additional substates. The "open" state was more populated than the "semi-open" or "closed" states. In addition to free energies and barriers within the landscape, we characterized the average transition state passage time of ≈0.3 μs and reversible substrate and product binding. Human PGK in a dilute solution simulation shows a transition directly from the open to closed states, in agreement with previous SAXS experiments, suggesting that the cell-like model environment promotes stability of the human PGK semi-open state. Yeast PGK also sampled three metastable states within the cytoplasm model, with the closed state favored in our simulation.

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

The investigation and understanding of the folding mechanism of multidomain proteins is still a challenge in structural biology. The use of single-molecule Förster resonance energy transfer offers a unique tool to map conformational changes within the protein structure. Here, we present a study following denaturant-induced unfolding transitions of yeast phosphoglycerate kinase by mapping several inter- and intradomain distances of this two-domain protein, exhibiting a quite heterogeneous behavior. On the one hand, the development of the interdomain distance during the unfolding transition suggests a classical two-state unfolding behavior. On the other hand, the behavior of some intradomain distances indicates the formation of a compact and transient molten globule intermediate state. Furthermore, different intradomain distances measured within the same domain show pronounced differences in their unfolding behavior, underlining the fact that the choice of dye attachment positions within the polypeptide chain has a substantial impact on which unfolding properties are observed by single-molecule Förster resonance energy transfer measurements. Our results suggest that, to fully characterize the complex folding and unfolding mechanism of multidomain proteins, it is necessary to monitor multiple intra- and interdomain distances because a single reporter can lead to a misleading, partial, or oversimplified interpretation.

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

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.

MgF3- and AlF4- transition state analogue complexes of yeast phosphoglycerate kinase.

The phospho-transfer mechanism of yeast phosphoglycerate kinase (PGK) has been probed through formation of trifluoromagnesate (MgF3-) and tetrafluoroaluminate (AlF4-) transition state analogue complexes and analyzed using 19F, 1H waterLOGSY and 1H chemical shift perturbation NMR spectroscopy. We observed the first 19F NMR spectroscopic evidence for the formation of metal fluoride transition state analogues of yeast PGK and also observed significant changes to proton chemical shifts of PGK in the presence, but not in the absence, of fluoride upon titration of ligands, providing indirect evidence of the formation of a closed ternary transition state. WaterLOGSY NMR spectroscopy experiments using an uncompetitive model were used in an attempt to measure ligand binding affinities within the transition state analogue complexes.

Coiled-Coil Probes Identified the Unfolding Pathway of Yeast Phosphoglycerate Kinase

Large multi-domain proteins which have complicated reacting interfaces and dominate the proteomes of life remain mysterious regarding to their folding behaviors and mechanisms. Comprehensive studies towards the understanding of the fundamentals of protein folding mechanisms tend to bias small single-domain monomeric proteins, possibly due to their simplicity and reversible refolding capabilities. Yet the understanding of protein folding behaviors acquired from studying small single-domain proteins are not adequate to generate an accurate extrapolation for predicting the folding mechanisms of proteins that contain more than one domain. Here we present the characterization and interpretation of the unfolding pathway of a model large multi-domain protein yeast phosphoglycerate kinase (PGK) using single-molecule force spectroscopy (SMFS) and insertion of the antiparallel coiled-coil (CC) polypeptide probe we developed previously. When placed into different loop regions inside of yeast PGK, the CC probe affected different portion of the unfolding profile. Statistical comparison between the force-extension (FE) curves of multiple CC inserted yeast PGK's and the wildtype yeast PGK provided insights into figuring out the effects of conformational changes in domains and across the interfaces between domains on the unfolding pathway of the entire yeast PGK protein. Together with coarse-grain simulation modeling of the unfolding pathway, the sequence of the unfolding events occurred along the unfolding pathway of yeast PGK is precisely determined.

Minima and Barriers on the Pressure-Temperature Free Energy Landscape of Phosphoglycerate Kinase

Proteins can exhibit strikingly different free energy landscapes depending on which thermodynamic variable is manipulated to study folding. We have investigated the relationship between temperature- and pressure-induced denaturation of yeast phosphoglycerate kinase (PGK). Thermodynamically, the pressure-temperature phase diagram of PGK seems standard: with a slight deviation from two-state behavior at low temperatures, a predicted elliptical shape of stability emerges. Kinetically, the behavior is much more complex: although a temperature perturbation of the energy landscape of PGK reveals a multitude of barriers, pressure-jump experiments spanning timescales from microsecond to hours showed only one predominant several-minute-long kinetic phase. These observations may indicate that under pressure, the smaller kinetic barriers of PGK are smoothed out by the large positive activation volume of the transition state.

Temporal Variation of a Protein Folding Energy Landscape in the Cell

Chemical reaction rate coefficients and free energies are usually time-independent quantities. Protein folding in vitro is one such reaction with a fixed energy landscape. However, in the milieu of the cell, the energy landscape can be modulated in space and time by fluctuations in the intracellular environment such as cytoskeletal rearrangements, changes in biomolecule concentrations, and large scale cellular reorganization. We studied the time dependence of the folding landscape of a FRET-labeled enzyme, yeast phosphoglycerate kinase (PGK-FRET). Living U2OS cells served as our test tube, and the mammalian cell cycle, a process strictly regulated in time, served as our clock. We found that both the rate of folding and the thermodynamic stability of PGK-FRET are cell cycle-dependent. We also assayed folding rates of PGK-FRET in spatial proximity to and far away from mitotic chromosomes. Our results show that expedited folding in DNA-rich regions cannot account for the faster rate of PGK-FRET folding in mitotic cells.

Deciphering Protein Stability in Cells

Deciphering Protein Stability in Cells

Transformation ofCandida guilliermondiiwild-type strains using theStaphylococcus aureusMRSA 252blegene as a phleomycin-resistant marker

We designed an efficient transformation system for Candida guilliermondii wild-type strains. We demonstrated that the Staphylococcus aureus MRSA 252 ble coding sequence placed under the control of the yeast phosphoglycerate kinase gene transcription-regulating regions confers phleomycin resistance to transformed C.guilliermondii cells. To illustrate the potential of this drug-resistant cassette, we carried out the disruption of the C.guilliermondii ADE2 gene. This new dominant selectable marker represents a powerful tool to study the function of various genes in this yeast of clinical and biotechnological interest.

Protein Folding across the Cell Cycle

The mammalian cell cycle is the biological pathway through which cells grow and divide. During this physically dramatic process the cell doubles in size, duplicates its genetic material, dissolves its nucleus, and ultimately divides into two genetically identical daughter cells. Due in part to the fundamental role of cell cycle abnormalities to cancer pathogenesis, cell cycle research remains a very active topic in the literature. Yet, there is a comparatively very little work exploring the influence of the cell cycle on protein biophysics. Considering the immense intracellular physical changes the cell undergoes during the cell cycle as well as the established sensitivity of protein folding to the intracellular environment, we aim to determine the in vivo protein folding trends as the cell progresses through the stages of the cell cycle. With Fast Relaxation Imaging (FReI), a fluorescence microscopy system for measuring in vivo protein folding, we explore the temperature induced folding thermodynamics and kinetics of a FRET-labeled model protein, yeast phosphoglycerate-kinase (PGK), in living U2OS cells arrested in the prometaphase stage of mitosis and the G1 stage of interphase. We see that PGK is more stable in mitotic cells compared to interphase cells, suggesting that the protein folding environment significantly changes as the cell progresses through the cell cycle.

Comparing the Folding and Misfolding Energy Landscapes of Phosphoglycerate Kinase

Partitioning of polypeptides between protein folding and amyloid formation is of outstanding pathophysiological importance. Using yeast phosphoglycerate kinase as model, here we identify the features of the energy landscape that decide the fate of the protein: folding or amyloidogenesis. Structure formation was initiated from the acid-unfolded state, and monitored by fluorescence from 10 ms to 20 days. Solvent conditions were gradually shifted between folding and amyloidogenesis, and the properties of the energy landscape governing structure formation were reconstructed. A gradual transition of the energy landscape between folding and amyloid formation was observed. In the early steps of both folding and misfolding, the protein searches through a hierarchically structured energy landscape to form a molten globule in a few seconds. Depending on the conditions, this intermediate either folds to the native state in a few minutes, or forms amyloid fibers in several days. As conditions are changed from folding to misfolding, the barrier separating the molten globule and native states increases, although the barrier to the amyloid does not change. In the meantime, the native state also becomes more unstable and the amyloid more stable. We conclude that the lower region of the energy landscape determines the final protein structure.

Comparing the Folding and Misfolding Energy Landscapes of Phosphoglycerate Kinase

Understanding the sequence specific partitioning of polypeptides between protein folding and amyloid formation is of outstanding physiological and pathological importance. Using yeast phosphoglycerate kinase as model, here we identify the features of the energy landscape that decide the fate of the protein: folding or amyloidogenesis. Structure formation was initiated from the acid-unfolded state by stopped-flow or manual mixing, and monitored by tryptophan and thioflavin T fluorescence from 1 ms to 10 days. Solvent conditions were gradually shifted between folding and amyloidogenesis and the properties of the energy landscape governing structure formation were reconstructed. A continuous transition of the energy landscape between folding and amyloid formation was observed. In the early steps of both folding and misfolding, the protein searches through a hierarchically structured energy landscape to form a molten globule on the seconds timescale. From this intermediate structure, folding to the native structure happens in a cross-barrier step in a few minutes, while amyloidogenesis progresses much slower, on the days and weeks timescale. As conditions are changed from folding to misfolding, formation of the native structure slows down indicating the increase of the barrier separating the molten globule and native states. In the meantime, the native state becomes more unstable as well. Amyloid formation is only observed among solvent conditions where folding is absent.

Structure, Function and Folding of Phosphoglycerate Kinase are Strongly Perturbed by Macromolecular Crowding

We combine experiment and computer simulation to show how macromolecular crowding dramatically affects the structure, function and folding landscape of phosphoglycerate kinase (PGK). Fluorescence labeling shows that compact states of yeast PGK are populated as the amount of crowding agents (Ficoll 70) increases. Coarse-grained molecular simulations reveal three compact ensembles: C (crystal structure), CC (collapsed crystal) and Sph (spherical compact). With an adjustment for viscosity, crowded wild type PGK and fluorescent PGK are about 15 times or more active in 200 mg/ml Ficoll than in aqueous solution. Our results suggest a new solution to the classic problem of how the ADP and diphosphoglycerate binding sites of PGK come together to make ATP: rather than undergoing a hinge motion, the ADP and substrate sites are already located in proximity under crowded conditions that mimic the in vivo conditions under which the enzyme actually operates. We also examine T-jump unfolding of PGK as a function of crowding experimentally. We uncover a non-monotonic folding relaxation time vs. Ficoll concentration. Theory and modeling explain why an optimum concentration exists for fastest folding. Below the optimum, folding slows down because the unfolded state is stabilized relative to the transition state. Above the optimum, folding slows down because of increased viscosity.

Structure, function, and folding of phosphoglycerate kinase are strongly perturbed by macromolecular crowding

We combine experiment and computer simulation to show how macromolecular crowding dramatically affects the structure, function, and folding landscape of phosphoglycerate kinase (PGK). Fluorescence labeling shows that compact states of yeast PGK are populated as the amount of crowding agents (Ficoll 70) increases. Coarse-grained molecular simulations reveal three compact ensembles: C (crystal structure), CC (collapsed crystal), and Sph (spherical compact). With an adjustment for viscosity, crowded wild-type PGK and fluorescent PGK are about 15 times or more active in 200mg/ml Ficoll than in aqueous solution. Our results suggest a previously undescribed solution to the classic problem of how the ADP and diphosphoglycerate binding sites of PGK come together to make ATP: Rather than undergoing a hinge motion, the ADP and substrate sites are already located in proximity under crowded conditions that mimic the in vivo conditions under which the enzyme actually operates. We also examine T-jump unfolding of PGK as a function of crowding experimentally. We uncover a nonmonotonic folding relaxation time vs. Ficoll concentration. Theory and modeling explain why an optimum concentration exists for fastest folding. Below the optimum, folding slows down because the unfolded state is stabilized relative to the transition state. Above the optimum, folding slows down because of increased viscosity.

Using Difference Infrared Spectroscopy to Investigate the Effects of pH on PGK-Substrate Complexes

Yeast phosphoglycerate kinase catalyzes the reversible phosphate transfer in the reaction: ADP +1,3-bis-phosphoglycerate ↔ ATP + 3-phosphoglycerate. Prior research indicates a hinge-bending mechanism occurs during catalysis to bring the substrates into closer proximity. Domain closure is only initiated in ternary complexes, in which both substrates are simultaneously bound to the enzyme. The activity and conformation of PGK is directly influenced by substrate and salt concentrations as well as pH.

Thermodynamics and Kinetics of the Pressure Unfolding of Phosphoglycerate Kinase

Due to the relationship between compressibility and volume fluctuations, high-pressure studies provide vital insight into protein dynamics and function. Most high-pressure experiments were performed on small and fast folding proteins or model peptides. Here we show that a detailed kinetic study is necessary to extract reliable information from the high-pressure-induced structural conversion of large, slowly folding proteins. The pressure-jump unfolding kinetics of yeast phosphoglycerate kinase was recorded at pressures between 50 and 150 MPa. The time dependence of the conformational state of the protein was followed by tryptophan fluorescence measurements from 30 s to 2 h. The observed changes were described by a three-state model, and the volume change and the activation volume as well as the midpoint pressure of the transitions between the folded, intermediate, and unfolded states were determined. An interesting feature of the pressure unfolding of phosphoglycerate kinase was that the unfolding process speeds up with increasing pressure, which is the consequence of negative activation volumes for the folded --> intermediate, intermediate --> unfolded, and unfolded --> intermediate transitions.

Method to Characterize the Global Dynamics of Proteins by Temperature Dependent Phosphorescence Lifetime Measurements. Application for the Allosteric Effect in Hemoglobin

At the glass transition temperature of proteins, large scale, anharmonic motions of the conformation become activated. It has been shown that in this temperature range the phosphorescence lifetime of Trp residues that is long (several s) at cryogenic temperatures become efficiently quenched. We report the elaboration of a systematic method based on this observation with the purpose to use this effect for characterizing the global conformational dynamics of proteins. In this method, special conditions were determined under which the onset temperature of phosphorescence quenching can be clearly separated from the slaving effect of fluctuations activated in the solvent, and the relevance of this temperature for the global dynamics of the protein has been verified by parallel experiments on human alpha-oxyFe-betaZn-Hemoglobin, w.t. horse heart Myoglobin and Trp mutant forms of a two-domain protein, yeast phosphoglycerate kinase. The method was used to characterize the role of global dynamics in the allosteric response of human hemoglobin to the binding of effectors as Cl, IHP, DPG, BZF. The quenching effect showed a two state behavior with a characteristic onset temperature sensitive to the presence and quality of allosteric effectors. The data points were fit by a simple two state model and the thermodynamic parameters - enthalpy and entropy changes characteristic for the quenching effect were discussed in terms of oxygen hopping models. The study on hemoglobin verifies the concept that in allosteric regulation, the global dynamics of the protein tetramer plays important role.Acknowledgements: the authors thank Prof. T. Yonetani of University of Pennsylvania for kindly providing Zn-PP- substituted human Hb for the studies. J. Fidy thank the Hung. Ministry of Health for support by grant no. ETT 512/2006.

Using Difference Infrared Spectroscopy to Investigate the Effects of pH on PGK-Substrate Complexes

Yeast phosphoglycerate kinase catalyzes the reversible phosphate transfer in the reaction: ADP +1,3-bis-phosphoglycerate ↔ ATP + 3-phosphoglycerate. Prior research indicates a hinge-bending mechanism occurs during catalysis to bring the substrates into closer proximity. Domain closure is only initiated in ternary complexes, in which both substrates are simultaneously bound to the enzyme. The activity and conformation of PGK is directly influenced by substrate and salt concentrations as well as pH.

Thermodynamic analysis of the nondenaturational conformational change of baker’s yeast phosphoglycerate kinase at 24 °C

A plot of the Gibbs free energy of unfolding vs. temperature is calculated for baker’s yeast phosphoglycerate kinase in 150 mM sodium phosphate (pH = 7.0) from a combination of reversible differential scanning calorimetry measurements and isothermal guanidine hydrochloride titrations. The stability curve reveals the existence of two stable, folded conformers with an abrupt conformational transition occurring at 24 °C. The transition state thermodynamics for the low- to high-temperature conformational change are calculated from slow-scan-rate differential scanning calorimetry measurements where it is found that the free energy barrier for the conversion is 90 kJ/mol and the transition state possesses a significant unfolding quality. This analysis also confirms a nondenaturational conformational transition at 24 °C. The data therefore suggest that X-ray structures obtained from crystals grown below this temperature may differ considerably from the physiological structure and that the two conformers are not readily interconverted.

Engineering volatile thiol release in Saccharomyces cerevisiae for improved wine aroma

Volatile thiols, such as 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), are among the most potent aroma compounds found in wine and can have a significant effect on wine quality and consumer preferences. At optimal concentrations in wine, these compounds impart flavours of passionfruit, grapefruit, gooseberry, blackcurrant, lychee, guava and box hedge. The enzymatic release of aromatic thiols from grape-derived, non-volatile cysteinylated precursors (Cys-4MMP and Cys-3MH) and the further modification thereof (conversion of 3MH into 3MHA) during fermentation, enhance the varietal characters of wines such as Sauvignon Blanc. Wine yeast strains have limited and varying capacities to produce aroma-enhancing thiols from their non-volatile counterparts in grape juice. Even under optimal fermentation conditions, the most efficient thiol-releasing Saccharomyces cerevisiae wine strain known realizes less than 5% of the thiol-related flavour potential of grape juice. The objective of this study was to develop a wine yeast able to unleash the untapped thiol aromas in grape juice during winemaking. To achieve this goal, the Escherichia coli tnaA gene, encoding a tryptophanase with strong cysteine-beta-lyase activity, was cloned and overexpressed in a commercial wine yeast strain under the control of the regulatory sequences of the yeast phosphoglycerate kinase I gene (PGK1). This modified strain expressing carbon-sulphur lyase activity released up to 25 times more 4MMP and 3MH in model ferments than the control host strain. Wines produced with the engineered strain displayed an intense passionfruit aroma. This yeast offers the potential to enhance the varietal aromas of wines to predetermined market specifications.