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.

A Trypanosoma cruzi phosphoglycerate kinase isoform with a Per-Arnt-Sim domain acts as a possible sensor for intracellular conditions.

Per-ARNT-Sim (PAS) domains constitute a family of domains present in a wide variety of prokaryotic and eukaryotic organisms. They form part of the structure of various proteins involved in diverse cellular processes. Regulation of enzymatic activity and adaptation to environmental conditions, by binding small ligands, are the main functions attributed to PAS-containing proteins. Recently, genes for a diverse set of proteins with a PAS domain were identified in the genomes of several protists belonging to the group of kinetoplastids, however, until now few of these proteins have been characterized. In this work, we characterize a phosphoglycerate kinase containing a PAS domain present in Trypanosoma cruzi (TcPAS-PGK). This PGK isoform is an active enzyme of 58 kDa with a PAS domain located at its N-terminal end. We identified the protein's localization within glycosomes of the epimastigote form of the parasite by differential centrifugation and selective permeabilization of its membranes with digitonin, as well as in an enriched mitochondrial fraction. Heterologous expression systems were developed for the protein with the N-terminal PAS domain (PAS-PGKc) and without it (PAS-PGKt), and the substrate affinities of both forms of the protein were determined. The enzyme does not exhibit standard Michaelis-Menten kinetics. When evaluating the dependence of the specific activity of the recombinant PAS-PGK on the concentration of its substrates 3-phosphoglycerate (3PGA) and ATP, two peaks of maximal activity were found for the complete enzyme with the PAS domain and a single peak for the enzyme without the domain. Km values measured for 3PGA were 219 ± 26 and 8.8 ± 1.3 µM, and for ATP 291 ± 15 and 38 ± 2.2 µM, for the first peak of PAS-PGKc and for PAS-PGKt, respectively, whereas for the second PAS-PGKc peak values of approximately 1.1-1.2 mM were estimated for both substrates. Both recombinant proteins show inhibition by high concentrations of their substrates, ATP and 3PGA. The presence of hemin and FAD exerts a stimulatory effect on PAS-PGKc, increasing the specific activity by up to 55%. This stimulation is not observed in the absence of the PAS domain. It strongly suggests that the PAS domain has an important function in vivo in T. cruzi in the modulation of the catalytic activity of this PGK isoform. In addition, the PAS-PGK through its PAS and PGK domains could act as a sensor for intracellular conditions in the parasite to adjust its intermediary metabolism.

Synergistic effect of conformational changes in phosphoglycerate kinase 1 product release.

In the glycolysis pathway, phosphoglycerate kinase 1 (PGK1) transfers one phosphoryl-group from 1,3-diphosphoglycerate (1,3BPG) to ADP to product 3-phosphoglycerate (3PG) and ATP. The catalytic process is accompanied with the conversion between the open conformation and the closed conformation of PGK1. However, the dynamic collaboration mechanism between the PGK1 conformation transition and the products releasing process remains poorly understood. Here using molecular dynamics simulations combined with molecular mechanics generalized born surface area (MM/GBSA) analysis, we demonstrated that PGK1 in the closed conformation first releases the product ATP to reach a semi-open conformation, and releases the product 3PG to achieve the full open conformation, which could accept new substrates ADP and 1,3BPG for the next cycle. It is noteworthy that the phosphorylation of PGK1 at T243 causes the loop region (residues L248-E260) flip outside the protein, and the phosphorylation of Y324 leads PGK1 become looser. Both modifications cause the exposure of the ADP/ATP binding site, which was beneficial for the substrates/products binding/releasing of PGK1. In addition, the other post translational modifications (PTMs) were also able to regulate the ligands binding/releasing with different effects. Our results revealed the dynamic cooperative molecular mechanism of PGK1 conformational transition with products releasing, as well as the influence of PTMs, which would contribute to the understanding of PGK1 substrates/products conversion process and the development of small molecule drugs targeting PGK1.Communicated by Ramaswamy H. Sarma.

Roles and mechanisms of phosphoglycerate kinase 1 in cancer.

Phosphoglycerate kinase 1 (PGK1) catalyzes the conversion of 1,3-bisphosphoglyceride (1,3-BPG) and ADP into 3-phosphate (3-PG) and ATP, which is a key process of glycolysis. PGK1 is considered a major regulator of various events, including one-carbon metabolism, serine biosynthesis and cell redox regulation. In the past decade, PGK1 has been found to be closely associated with various malignancies, making it a potential therapeutic target. PGK1 is involved in a series of biological processes related to tumorigenesis through post-translational modifications and various signaling pathways. PGK1 not only can participate in glucose metabolism but also acts as a protein kinase to participate in EMT, autophagy, angiogenesis, DNA replication and other processes related to tumor development. However, PGK1 also acts as a disulfide reductase to inhibit tumor by affecting angiogenesis. Exploring the structure, function and posttranslational modification of PGK1 will be helpful in further understanding the effect of metabolism on tumor progression. This manuscript reviews the role and mechanism of PGK1 in human malignancies, providing the theoretical basis for PGK1 as a possible clinical anticancer target.

Protein Stabilization by Alginate Binding and Suppression of Thermal Aggregation.

Polymers designed to stabilize proteins exploit direct interactions or crowding, but mechanisms underlying increased stability or reduced aggregation are rarely established. Alginate is widely used to encapsulate proteins for drug delivery and tissue regeneration despite limited knowledge of its impact on protein stability. Here, we present evidence that alginate can both increase protein folding stability and suppress the aggregation of unfolded protein through direct interactions without crowding. We used a fluorescence-based conformational reporter of two proteins, the metabolic protein phosphoglycerate kinase (PGK) and the hPin1 WW domain to monitor protein stability and aggregation as a function of temperature and the weight percent of alginate in solution. Alginate stabilizes PGK by up to 14.5 °C, but stabilization is highly protein-dependent, and the much smaller WW domain is stabilized by only 3.5 °C against thermal denaturation. Stabilization is greatest at low alginate weight percent and decreases at higher alginate concentrations. This trend cannot be explained by crowding, and ionic screening suggests that alginate stabilizes proteins through direct interactions with a significant electrostatic component. Alginate also strongly suppresses aggregation at high temperature by irreversibly associating with unfolded proteins and preventing refolding. Both the beneficial and negative impacts of alginate on protein stability and aggregation have important implications for practical applications.

A Code Within a Code: How Codons Fine-Tune Protein Folding in the Cell.

The genetic code sets the correspondence between the sequence of a given nucleotide triplet in an mRNA molecule, called a codon, and the amino acid that is added to the growing polypeptide chain during protein synthesis. With four bases (A, G, U, and C), there are 64 possible triplet codons: 61 sense codons (encoding amino acids) and 3 nonsense codons (so-called, stop codons that define termination of translation). In most organisms, there are 20 common/standard amino acids used in protein synthesis; thus, the genetic code is redundant with most amino acids (with the exception of Met and Trp) are being encoded by more than one (synonymous) codon. Synonymous codons were initially presumed to have entirely equivalent functions, however, the finding that synonymous codons are not present at equal frequencies in mRNA suggested that the specific codon choice might have functional implications beyond coding for amino acid. Observation of nonequivalent use of codons in mRNAs implied a possibility of the existence of auxiliary information in the genetic code. Indeed, it has been found that genetic code contains several layers of such additional information and that synonymous codons are strategically placed within mRNAs to ensure a particular translation kinetics facilitating and fine-tuning co-translational protein folding in the cell via step-wise/sequential structuring of distinct regions of the polypeptide chain emerging from the ribosome at different points in time. This review summarizes key findings in the field that have identified the role of synonymous codons and their usage in protein folding in the cell.

Substrates Modulate Charge-Reorganization Allosteric Effects in Protein-Protein Association.

Protein function may be modulated by an event occurring far away from the functional site, a phenomenon termed allostery. While classically allostery involves conformational changes, we recently observed that charge redistribution within an antibody can also lead to an allosteric effect, modulating the kinetics of binding to target antigen. In the present work, we study the association of a polyhistidine tagged enzyme (phosphoglycerate kinase, PGK) to surface-immobilized anti-His antibodies, finding a significant Charge-Reorganization Allostery (CRA) effect. We further observe that PGK's negatively charged nucleotide substrates modulate CRA substantially, even though they bind far away from the His-tag-antibody interaction interface. In particular, binding of ATP reduces CRA by more than 50%. The results indicate that CRA is affected by the binding of charged molecules to a protein and provide further insight into the significant role that charge redistribution can play in protein function.

Phosphoglycerate kinase: structural aspects and functions, with special emphasis on the enzyme from Kinetoplastea.

Phosphoglycerate kinase (PGK) is a glycolytic enzyme that is well conserved among the three domains of life. PGK is usually a monomeric enzyme of about 45 kDa that catalyses one of the two ATP-producing reactions in the glycolytic pathway, through the conversion of 1,3-bisphosphoglycerate (1,3BPGA) to 3-phosphoglycerate (3PGA). It also participates in gluconeogenesis, catalysing the opposite reaction to produce 1,3BPGA and ADP. Like most other glycolytic enzymes, PGK has also been catalogued as a moonlighting protein, due to its involvement in different functions not associated with energy metabolism, which include pathogenesis, interaction with nucleic acids, tumorigenesis progression, cell death and viral replication. In this review, we have highlighted the overall aspects of this enzyme, such as its structure, reaction kinetics, activity regulation and possible moonlighting functions in different protistan organisms, especially both free-living and parasitic Kinetoplastea. Our analysis of the genomes of different kinetoplastids revealed the presence of open-reading frames (ORFs) for multiple PGK isoforms in several species. Some of these ORFs code for unusually large PGKs. The products appear to contain additional structural domains fused to the PGK domain. A striking aspect is that some of these PGK isoforms are predicted to be catalytically inactive enzymes or 'dead' enzymes. The roles of PGKs in kinetoplastid parasites are analysed, and the apparent significance of the PGK gene duplication that gave rise to the different isoforms and their expression in Trypanosoma cruzi is discussed.

Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells.

The dynamic cytoskeletal network of microtubules and actin filaments can be disassembled by drugs. Cytoskeletal drugs work by perturbing the monomer-polymer equilibrium, thus changing the size and number of macromolecular crowders inside cells. Changes in both crowding and nonspecific surface interactions ("sticking") following cytoskeleton disassembly can affect the protein stability, structure, and function directly or indirectly by changing the fluidity of the cytoplasm and altering the crowding and sticking of other macromolecules in the cytoplasm. The effect of cytoskeleton disassembly on protein energy landscapes inside cells has yet to be observed. Here we have measured the effect of several cytoskeletal drugs on the folding energy landscape of two FRET-labeled proteins with different in vitro sensitivities to macromolecular crowding. Phosphoglycerate kinase (PGK) was previously shown to be more sensitive to crowding, whereas variable major protein-like sequence expressed (VlsE) was previously shown to be more sensitive to sticking. The in-cell effects of drugs that depolymerize either actin filaments (cytochalasin D and latrunculin B) or microtubules (nocodazole and vinblastine) were compared. The crowding sensor protein CrH2-FRET verified that cytoskeletal drugs decrease the extent of crowding inside cells despite also reducing the overall cell volume. The decreased compactness and folding stability of PGK could be explained by the decreased extent of crowding induced by these drugs. VlsE's opposite response to the drugs shows that depolymerization of the cytoskeleton also changes sticking in the cellular milieu. Our results demonstrate that perturbation of the monomer-polymer cytoskeletal equilibrium, for example, during natural cell migration or stresses from drug treatment, has off-target effects on the energy landscapes of proteins in the cell.

Perturbation of phosphoglycerate kinase 1 (PGK1) only marginally affects glycolysis in cancer cells.

Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell glycolysis is unclear. Here, we developed an enzyme assay to measure the kinetic parameters of the PGK1-catalyzed forward reaction. The Km values for 1,3-bisphosphoglyceric acid (1,3-BPG, the forward reaction substrate) were 4.36 µm (yeast PGK1) and 6.86 µm (human PKG1). The Km values for 3-phosphoglycerate (3-PG, the reverse reaction substrate and a serine precursor) were 146 µm (yeast PGK1) and 186 µm (human PGK1). The Vmax of the forward reaction was about 3.5- and 5.8-fold higher than that of the reverse reaction for the human and yeast enzymes, respectively. Consistently, the intracellular steady-state concentrations of 3-PG were between 180 and 550 µm in cancer cells, providing a basis for glycolysis to shuttle 3-PG to the serine synthesis pathway. Using siRNA-mediated PGK1-specific knockdown in five cancer cell lines derived from different tissues, along with titration of PGK1 in a cell-free glycolysis system, we found that the perturbation of PGK1 had no effect or only marginal effects on the glucose consumption and lactate generation. The PGK1 knockdown increased the concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and 1,3-BPG in nearly equal proportions, controlled by the kinetic and thermodynamic states of glycolysis. We conclude that perturbation of PGK1 in cancer cells insignificantly affects the conversion of glucose to lactate in glycolysis.

O-GlcNAcylation of PGK1 coordinates glycolysis and TCA cycle to promote tumor growth.

Many cancer cells display enhanced glycolysis and suppressed mitochondrial metabolism. This phenomenon, known as the Warburg effect, is critical for tumor development. However, how cancer cells coordinate glucose metabolism through glycolysis and the mitochondrial tricarboxylic acid (TCA) cycle is largely unknown. We demonstrate here that phosphoglycerate kinase 1 (PGK1), the first ATP-producing enzyme in glycolysis, is reversibly and dynamically modified with O-linked N-acetylglucosamine (O-GlcNAc) at threonine 255 (T255). O-GlcNAcylation activates PGK1 activity to enhance lactate production, and simultaneously induces PGK1 translocation into mitochondria. Inside mitochondria, PGK1 acts as a kinase to inhibit pyruvate dehydrogenase (PDH) complex to reduce oxidative phosphorylation. Blocking T255 O-GlcNAcylation of PGK1 decreases colon cancer cell proliferation, suppresses glycolysis, enhances the TCA cycle, and inhibits tumor growth in xenograft models. Furthermore, PGK1 O-GlcNAcylation levels are elevated in human colon cancers. This study highlights O-GlcNAcylation as an important signal for coordinating glycolysis and the TCA cycle to promote tumorigenesis.

Characterization of filarial phosphoglycerate kinase.

Phosphoglycerate kinase (PGK) is a key enzyme of glycolysis which also acts as a mediator of DNA replication and repair in the nucleus. We have cloned and expressed PGK in Brugia malayi. The rBmPGK was found to be 415 amino acid residues long having 45 kDa subunit molecular weight. This enzyme was also identified in different life stages of bovine filarial parasite Setaria cervi. The enzyme activity was highest in microfilarial stage followed by adult female and male as also shown by real time PCR in the present study. Further using BmPGK primers the cDNA prepared from S. cervi was amplified and sequenced which showed 100% homology with Brugia malayi PGK. B. malayi and S. cervi, PGK consists of conserved calmodulin binding domain (CaMBD) having 21 amino acids. In the present study we have shown the CaMBD binds to calcium-calmodulin and regulates its activity. The binding of calmodulin (CaM) with CaMBD was confirmed using calmodulin agarose binding pull down assay, which showed that the rBmPGK binds to CaM agarose-calcium dependent manner. The effect of CaM-Ca2+on the activity of rBmPGK was studied at different concentration of CaM (0.01-5.0 µM) and calcium chloride (0.01-100 µM). The rBmPGK was activated up to 85% in the presence of CaM at 1 µM and 10 µM concentration of CaCl2. Interestingly this activation was abrogated by metal chelator EDTA. Similar results were shown in case of Setaria cervi PGK. A significant increase (90 ±â€¯10) % in ScPGK activity was observed in the presence of CaM and CaCl2 at 1.0 µM and 1.0 mM respectively, further increase in the conc. of CaCl2, the activity of ScPGK was found to be decreased like rBmPGK. Bioinformatics studies have also confirmed the interaction between CaMBD and CaM which showed CaM interacted to Phe 206, Gln 220, Arg 223 and Asn 224 of rBmPGK CaM binding domain. On the basis of these findings, it has been suggested that the activity of filarial PGK could be regulated in cells by Ca2+-CaM depending upon the concentration of calcium. To the best of our knowledge this is first report in filarial parasite.

A Novel Pyrazolopyrimidine Ligand of Human PGK1 and Stress Sensor DJ1 Modulates the Shelterin Complex and Telomere Length Regulation.

Telomere signaling and metabolic dysfunction are hallmarks of cell aging. New agents targeting these processes might provide therapeutic opportunities, including chemoprevention strategies against cancer predisposition. We report identification and characterization of a pyrazolopyrimidine compound series identified from screens focused on cell immortality and whose targets are glycolytic kinase PGK1 and oxidative stress sensor DJ1. We performed structure-activity studies on the series to develop a photoaffinity probe to deconvolute the cellular targets. In vitro binding and structural analyses confirmed these targets, suggesting that PGK1/DJ1 interact, which we confirmed by immunoprecipitation. Glucose homeostasis and oxidative stress are linked to telomere signaling and exemplar compound CRT0063465 blocked hypoglycemic telomere shortening. Intriguingly, PGK1 and DJ1 bind to TRF2 and telomeric DNA. Compound treatment modulates these interactions and also affects Shelterin complex composition, while conferring cellular protection from cytotoxicity due to bleomycin and desferroxamine. These results demonstrate therapeutic potential of the compound series.

Characterization and role of PGK from Litopenaeus vannamei in WSSV infection.

Phosphoglycerate kinase (EC 2.7.2.3, PGK) catalyses the reversible transfer of a phosphate group from 1,3-diphosphoglyceric acid and ADP to produce 3-phosphoglyceric acid and ATP, which represents the initial production of ATP during glycolysis; therefore, PGK is a key enzyme in the energy metabolism. To study the role of PGK in the resistance to WSSV infection in shrimp, the full-length cDNA of the PGK gene (LvPGK) from Litopenaeus vannamei was obtained by using homology cloning and RACE amplification. The tissue distribution of LvPGK and its expression changes in the main immune tissues after WSSV stimulation were obtained by quantitative real-time PCR. Furthermore, RNA interference (RNAi) was used to study the role of LvPGK in shrimp defending against WSSV infection. The results showed that the full-length cDNA sequence of LvPGK was 1855 bp, contained a 1248 bp open reading frame (ORF) encoding 415 amino acids, and included a conserved PGK domain. LvPGK presented ubiquitous expression in most examined tissues, with the most predominant expression in the muscle and the weakest expression in the intestine. LvPGK transcripts could be induced in the hemocytes and hepatopancreas by injection with WSSV. Both the replication of WSSV and the shrimp cumulative mortality decreased significantly after LvPGK knockdown (P < 0.01). After challenging LvPGK RNAi shrimp with WSSV, the concentration of glucose in the hepatopancreas and muscle tissue did not show significant change; however, the content of pyruvate and lactate decreased significantly (P < 0.05). Moreover, significant decreases in the expression levels of crustin, ALF1, ALF2 and ALF3 were also detected. The results suggested that LvPGK might be involved in WSSV replication by increasing host aerobic and anaerobic metabolism.

Structural determinants increasing flexibility confer cold adaptation in psychrophilic phosphoglycerate kinase.

Crystal structures of phosphoglycerate kinase (PGK) from the psychrophile Pseudomonas sp. TACII 18 have been determined at high resolution by X-ray crystallography methods and compared with mesophilic, thermophilic and hyperthermophilic counterparts. PGK is a two-domain enzyme undergoing large domain movements to catalyze the production of ATP from 1,3-biphosphoglycerate and ADP. Whereas the conformational dynamics sustaining the catalytic mechanism of this hinge-bending enzyme now seems rather clear, the determinants which underlie high catalytic efficiency at low temperatures of this psychrophilic PGK were unknown. The comparison of the three-dimensional structures shows that multiple (global and local) specific adaptations have been brought about by this enzyme. Together, these reside in an overall increased flexibility of the cold-adapted PGK thereby allowing a better accessibility to the active site, but also a potentially more disordered transition state of the psychrophilic enzyme, due to the destabilization of some catalytic residues.

PAS domain-containing phosphoglycerate kinase deficiency in Leishmania major results in increased autophagosome formation and cell death.

Per-Arnt-Sim (PAS) domains are structurally conserved and present in numerous proteins throughout all branches of the phylogenetic tree. Although PAS domain-containing proteins are major players for the adaptation to environmental stimuli in both prokaryotic and eukaryotic organisms, these types of proteins are still uncharacterized in the trypanosomatid parasites, Trypanosome and Leishmania In addition, PAS-containing phosphoglycerate kinase (PGK) protein is uncharacterized in the literature. Here, we report a PAS domain-containing PGK (LmPAS-PGK) in the unicellular pathogen Leishmania The modeled structure of N-terminal of this protein exhibits four antiparallel ß sheets centrally flanked by α helices, which is similar to the characteristic signature of PAS domain. Activity measurements suggest that acidic pH can directly stimulate PGK activity. Localization studies demonstrate that the protein is highly enriched in the glycosome and its presence can also be seen in the lysosome. Gene knockout, overexpression and complement studies suggest that LmPAS-PGK plays a fundamental role in cell survival through autophagy. Furthermore, the knockout cells display a marked decrease in virulence when host macrophage and BALB/c mice were infected with them. Our work begins to clarify how acidic pH-dependent ATP generation by PGK is likely to function in cellular adaptability of Leishmania.

Quantifying protein dynamics and stability in a living organism.

As an integral part of modern cell biology, fluorescence microscopy enables quantification of the stability and dynamics of fluorescence-labeled biomolecules inside cultured cells. However, obtaining time-resolved data from individual cells within a live vertebrate organism remains challenging. Here we demonstrate a customized pipeline that integrates meganuclease-mediated mosaic transformation with fluorescence-detected temperature-jump microscopy to probe dynamics and stability of endogenously expressed proteins in different tissues of living multicellular organisms.

Rab11-FIP2 suppressed tumor growth via regulation of PGK1 ubiquitination in non-small cell lung cancer.

Mounting evidence has shown that the Rab11-FIP2 has critical roles in cancer cell growth. However, the clinical significance of Rab11-FIP2 in Non-small cell lung cancer (NSCLC) remains to be fully elucidated. In this study, we investigated the expression of Rab11-FIP2 using immunohistochemistry in 150 patients with NSCLC. We found that its expression level in NSCLC was much lower than that in the corresponding adjacent normal tissues. The DNA methylation data revealed that Rab11-FIP2 were significantly hypermethylated in NSCLC. The methylation level in the gene body was negatively correlated with the expression level of Rab11-FIP2 in NSCLC. Furthermore, enforced expression of Rab11-FIP2 dramatically reduced cancer cell proliferation and tumorigenesis, indicating a tumor suppressor role of PGK1 in NSCLC progression. Mechanistic investigations showed that Rab11-FIP2 interacted with the glycolytic kinase PGK1 and promoted its ubiquitination in NSCLC cells, leading to inactivation of the oncogenic AKT/mTOR signaling pathway. Overall, our data indicate that reduced expression of Rab11-FIP2 by DNA hypermethylation plays an important role in NSCLC tumor growth.

Soluble Zwitterionic Poly(sulfobetaine) Destabilizes Proteins.

The widespread interest in neutral, water-soluble polymers such as poly(ethylene glycol) (PEG) and poly(zwitterions) such as poly(sulfobetaine) (pSB) for biomedical applications is due to their widely assumed low protein binding. Here we demonstrate that pSB chains in solution can interact with proteins directly. Moreover, pSB can reduce the thermal stability and increase the protein folding cooperativity relative to proteins in buffer or in PEG solutions. Polymer-dependent changes in the tryptophan fluorescence spectra of three structurally-distinct proteins reveal that soluble, 100 kDa pSB interacts directly with all three proteins and changes both the local polarity near tryptophan residues and the protein conformation. Thermal denaturation studies show that the protein melting temperatures decrease by as much as ∼1.9 °C per weight percent of polymer and that protein folding cooperativity increases by as much as ∼130 J mol-1 K-1 per weight percent of polymer. The exact extent of the changes is protein-dependent, as some proteins exhibit increased stability, whereas others experience decreased stability at high soluble pSB concentrations. These results suggest that pSB is not universally protein-repellent and that its efficacy in biotechnological applications will depend on the specific proteins used.

The phosphoglycerate kinase 1 variants found in carcinoma cells display different catalytic activity and conformational stability compared to the native enzyme.

Cancer cells are able to survive in difficult conditions, reprogramming their metabolism according to their requirements. Under hypoxic conditions they shift from oxidative phosphorylation to aerobic glycolysis, a behavior known as Warburg effect. In the last years, glycolytic enzymes have been identified as potential targets for alternative anticancer therapies. Recently, phosphoglycerate kinase 1 (PGK1), an ubiquitous enzyme expressed in all somatic cells that catalyzes the seventh step of glycolysis which consists of the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate to ADP, has been discovered to be overexpressed in many cancer types. Moreover, several somatic variants of PGK1 have been identified in tumors. In this study we analyzed the effect of the single nucleotide variants found in cancer tissues on the PGK1 structure and function. Our results clearly show that the variants display a decreased catalytic efficiency and/or thermodynamic stability and an altered local tertiary structure, as shown by the solved X-ray structures. The changes in the catalytic properties and in the stability of the PGK1 variants, mainly due to the local changes evidenced by the X-ray structures, suggest also changes in the functional role of PGK to support the biosynthetic need of the growing and proliferating tumour cells.