Jorge Babul - Academia.edu (original) (raw)

Papers by Jorge Babul

Research paper thumbnail of Effect of ATP on phosphofructokinase-2 from Escherichia coli. A mutant enzyme altered in the allosteric site for MgATP

Journal of Biological Chemistry, Sep 1, 1985

The activity of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2* wa... more The activity of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2* was measured over a wide range of Mg2' and ATP concentrations. MgATP2-inhibited only the Pfk-2 enzyme, with a degree of cooperativity of 1.5. This inhibition was relieved upon increasing the fructose-6-P concentration or by lowering the pH of the reaction mixture. Other nucleotides used as phosphate donors instead of ATP did not inhibit. MgATP2-was the true substrate for both enzymes and their K,,, values for this compound were not affected by an increase of the free Mg2+ concentration. However, free Mg2' partially relieved the MgATP2-inhibition of Pfk-2 under conditions where the ATP4-concentration was negligible, without changes in the degree of cooperativity. ATP4-acted as a strong competitive inhibitor of both Pfk-2 and Pfk-2* with respect to MgATP2-with Ki values of 10 and 8 WM, respectively. ADP, AMP, and CAMP did not prevent the MgATP2-inhibition of Pfk-2. These results suggest the presence of an allosteric site for MgATP2-in Pfk-2 responsible for the MgATP2-inhibition, which is altered in Pfk-2* as a consequence of the structural mutation.

Research paper thumbnail of A phosphofructokinase mutant of Escherichia coli altered in its allosteric properties impairs gluconeogenic growth

PubMed, Dec 1, 1985

The regulation of metabolic fluxes is accomplished by modulation of key enzyme catalyzed reaction... more The regulation of metabolic fluxes is accomplished by modulation of key enzyme catalyzed reactions. This modulation takes place partially through the control of the catalytic activity of enzymes labelled as regulatory enzymes. The kinetic behavior of many regulatory enzymes can be explained in terms of multiple binding sites for effector molecules. Of these, the ones that play their control over catalysis by binding at an allosteric site have been considered of much importance. Nevertheless, proof that the effects observed in vitro, are in fact responsible for the physiological regulation in vivo, is scarce. In this regard, mutant enzymes altered in their allosteric properties might be useful. This will be illustrated with an enzyme considered crucial for the regulation of carbohydrate metabolism, namely phosphofructokinase. We present here the comparison of some of the kinetic and structural properties of wild type phosphofructokinase-2 of E. coli and of a mutant form which impairs gluconeogenic growth, an indication of the significance of the in vivo regulation. The main differences between the enzymes are their kinetic reaction mechanism, inhibitability by ATP, and aggregation states in the presence of substrates and effectors. So far these differences support only speculations as to the mechanism of the gluconeogenic impairment observed in strains that contain the mutant enzyme, a few of which are offered.

Research paper thumbnail of Crystal structure of the phosphofructokinase-2 from Escherichia coli in complex with fructose-6-phosphate

Research paper thumbnail of Ensemble Switching of the DNA-Binding Domain of Human FoxP1

Biophysical Journal, 2021

Rett Syndrome (RTT) is a rare monogenic progressive neurodevelopmental disorder occurring in abou... more Rett Syndrome (RTT) is a rare monogenic progressive neurodevelopmental disorder occurring in about 1 in 10,000 live female births. RTT is usually diagnosed within the first two years of life and manifested by loss of hand skills, impaired mobility and speech, and development of stereotypical hand movements. RTT is caused by mutations in methyl CpG-binding protein 2 (MeCP2). Several years ago, we studied the most common missense mutations in MBD domain of MeCP2 both in silico and in vitro to reveal their effect on protein structure, stability and binding to DNA. A range of methods including molecular dynamics and free energy perturbation (FEP) were employed and the predictions were compared with circular dichroism and gel mobility shift assay measurements. The 13 mutations investigated at the time were T158M,

Research paper thumbnail of Structural Dynamics of the Heterodimerization Between the DNA-Binding Domains from Human FoxP1 and FoP2 Transcription Factors

Biophysical Journal, 2021

Forkhead box P (FoxP) are members of the human Fox family of transcription factors (FoxA-FoxS), i... more Forkhead box P (FoxP) are members of the human Fox family of transcription factors (FoxA-FoxS), involved in diverse processes such as intellectual and immune system regulation. The four FoxP subfamily members share the unique ability to dimerize via domain swapping using their DNAbinding domain. Specifically, we study FoxP1 and FoxP2 dimerization due to its impact on various human diseases. Both proteins share 88% sequence identity but have distinct dimerization properties, with K D values ranging from mM (FoxP1) to mM (FoxP2). However, the structural and energetical properties of heterodimerization and the role of the DNA-binding domain and its DNA ligand in this association are unknown. In this context, we integrated single-molecule FRET and biophysical studies to probe the FoxP1-FoxP2 heterodimerization in vitro, emphasizing the effect that the ligand DNA has on the heterodimerization and the heterodimer's structural dynamics. Biophysical assessment of the heterodimerization indicated that the K D ascertained is similar to the FoxP1 homodimerization, and therefore dramatically different from the FoxP2 homodimerization value. Singlemolecule FRET analysis of FoxP1 homodimer and heterodimer (FoxP1:-FoxP2) showed that the heterodimer increases the local dynamics and the accumulation of an expanded intermediate, suggesting that FoxP2 presumably has evolved to heterodimerize instead of homodimerizing. Finally, DNAbinding assays indicated that monomer:DNA complex decreases the heterodimerization affinity, whereas having no substantial effect in the heterodimer's stability. These findings highlight relevant aspects of the regulatory role of DNA in the adoption of heterodimers and, therefore, in their gene-related expression.

Research paper thumbnail of New visible and selective DNA staining method in gels with tetrazolium salts

Analytical Biochemistry, 2017

DNA staining in gels has historically been carried out using silver staining and fluorescent dyes... more DNA staining in gels has historically been carried out using silver staining and fluorescent dyes like ethidium bromide and SYBR Green I (SGI). Using fluorescent dyes allows recovery of the analyte, but requires instruments such as a transilluminator or fluorimeter to visualize the DNA. Here we described a new and simple method that allows DNA visualization to the naked eye by generating a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in SGI and nitro blue tetrazolium (NBT) solution that, when exposed to sunlight, produces a purple insoluble formazan precipitate that remains in the gel after exposure to light. A calibration curve made with a DNA standard established a detection limit of approximately 60 pg/band at 500 bp. Selectivity of this assay was determined using different biomolecules, demonstrating a high selectivity for DNA. Integrity and functionality of the DNA recovered from gels was determined by enzymatic cutting with a restriction enzyme and by transforming competent cells after the different staining methods, respectively. Our method showed the best performance among the dyes employed. Based on its specificity, low cost and its adequacy for field work, this new methodology has enormous potential benefits to research and industry.

Research paper thumbnail of Crystal structure of the Phosphofructokinase-2 mutant Y23D from Escherichia coli

Research paper thumbnail of The alkaline isomerization of lysine-modified ferricytochrome c

Biochimica et Biophysica Acta (BBA) - Protein Structure, 1975

Both totally guanidinated and totally trifluoroacetylated ferricytochromes c exhibit a pH-depende... more Both totally guanidinated and totally trifluoroacetylated ferricytochromes c exhibit a pH-dependent displacement of the methionine ligand having an apparent pK within 0.5 pH units of that of the native protein. We propose that the pH dependence of this reaction reflects the concentration of hydroxide anion required to displace the methionine ligand.

Research paper thumbnail of An Alteration in Phosphofructokinase 2 of Escherichia coli which Impairs Gluconeogenic Growth and Improves Growth on Sugars

European Journal of Biochemistry, 2005

Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is ... more Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is allosteric, and a minor isoenzyme, phosphofructokinase 2. The pfkB1 mutation is known to increase the amount of phosphofructokinase 2 and allow growth on sugars of mutants lacking phosphofructokinase 1; it does not affect growth on substances such as glycerol or lactate (i.e., 'gluconeogenic growth'). However, gluconeogenic growth is markedly impaired in strains with a different allele, pfkB1*. We show here that strains with pfkB1* contain an altered form of phosphofructokinase 2, called phosphofructokinase 2*, which has been purified. Phosphofructokinase 2* is cold labile and has slightly different kinetic characteristics from phosphofructokinase 2, which include being less sensitive to inhibition by fructose 1,6-bisphosphate. The Km for fructose 6-phosphate is low (about 5 X 10(-5) M) in both phosphofructokinase 2 and phosphofructokinase 2*. However, in strains lacking phosphofructokinase 1, a high level of phosphofructokinase 2 is associated with unusually high concentrations of hexose monophosphates during growth on glucose, while a strain with phosphofructokinase 2* instead of phosphofructokinase 2 grows more rapidly on glucose and contains lower levels of hexose monophosphates. In gluconeogenic conditions, by contrast, hexose monophosphate levels are normal in phosphofructokinase 2 strains, while the impaired growth of phosphofructokinase 2* strains is associated with high levels of fructose 2,6-bisphosphate and very low levels of hexose monophosphates. These results show that phosphofructokinase 2, as studied in vitro, should no longer be regarded as a 'non-allosteric' protein, a conclusion also reached by Kotlarz and Buc on the basis of different types of experiments [Eur. J. Biochem. 117, 569-574 (1981)]. The fact that mutational alteration of phosphofructokinase 2 allows more rapid growth on glucose but severely impairs gluconeogenic growth is an indication of the significance of the regulation in vivo. The more rapid growth of the mutant on glucose might be explained on the basis of decreased sensitivity to an inhibitor (possibly, but not necessarily, fructose 1,6-bisphosphate), although other models are possible. A variety of speculations are offered as to the mechanism of gluconeogenic impairment.

Research paper thumbnail of An in vitro model showing different rates of substrate cycle for phosphofructokinases of Escherichia coli with different kinetic properties

European Journal of Biochemistry, 1991

An in vitro assay model is introduced for the coupled assay of phosphofructokinase (PFK) and fruc... more An in vitro assay model is introduced for the coupled assay of phosphofructokinase (PFK) and fructose-bisphosphatase. The model is applied to the study of three PFK of Escherichia coli: two isoenzymes, phosphofructokinase-1 (PFK-1) and phosphofructokinase-2 (PFK-2), and a mutant form of phosphofructokinase-2 (PFK-2*). Results show that for a variety of conditions the PFK-1/fructose-bisphosphatase pair gives the lowest and the PFK-2*/fructose-bisphosphatase pair the highest rates of substrate cycle, with the PFK-2/fructose-bisphosphatase pair in an intermediate position. The effects of variables such as maximum activity ratios and MgATP concentration were explored. The possible role of MgATP in decreasing the futile cycle of the PFK-2/fructose-bisphosphatase pair is described. The results are discussed in terms of possible metabolic consequences of PFK-2* and of predictions of the model to be tested in vivo.

Research paper thumbnail of Are the Aerobic and Anaerobic Phosphofructokinases of Escherichia coli Different?

European Journal of Biochemistry, 1977

Phosphofructokinase has been purified from Escherichia coli strain K-12 grown in a glucoselimited... more Phosphofructokinase has been purified from Escherichia coli strain K-12 grown in a glucoselimited chemostat, both aerobically and anaerobically. The enzymes migrated together in polyacrylamide gel electrophoresis, had the same subunit size in denaturing (dodecylsulfate) gels (M , approx. 34000) and the same kinetic characteristics as described earlier for E. coli phosphofructokinase [e.g. Blangy e f al. (1968) J. Mol. Biol. 31, 13-35]: a sigmoid curve of velocity vs. fructose 6-phosphate concentration, activation by ADP, and inhibition by phosphoenolpyruvate. Findings [e.g. Doelle (1975) Eur. J. Biochem. 50, 335-3421 of quite different enzymes in aerobic and anaerobic cells were not confirmed.

Research paper thumbnail of An examination of the involvement of proline peptide isomerization in protein folding

Journal of Molecular Biology, 1978

Research paper thumbnail of Reversible unfolding of dimeric phosphofructokinase-2 from Escherichia coli reveals a dominant role of inter-subunit contacts for stability

FEBS Letters, 2009

Escherichia coli phosphofructokinase-2 (Pfk-2) is a homodimer whose subunits consist of a large d... more Escherichia coli phosphofructokinase-2 (Pfk-2) is a homodimer whose subunits consist of a large domain and an additional b-sheet that provides the interfacial contacts between the subunits, creating a b-barrel flattened-like structure with the adjacent subunit's b-sheet. To determine how the structural organization of Pfk-2 determines its stability, the reversible unfolding of the enzyme was characterized under equilibrium conditions by enzymatic activity, circular dichroism, fluorescence and hydrodynamic measurements. Pfk-2 undergoes a cooperative unfolding/dissociation process with the accumulation of an expanded and unstructured monomeric intermediate with a marginal stability and a large solvent accessibility with respect to the native dimer.

Research paper thumbnail of The existence of heme-protein coordinate-covalent bonds in denaturing solvents

Research paper thumbnail of Unfolding Pathway of the Dimeric and Tetrameric Forms of Phosphofructokinase-2 from Escherichia coli †

Biochemistry, 2007

Escherichia coli phosphofructokinase-2 (Pfk-2) is an oligomeric enzyme characterized by two kinds... more Escherichia coli phosphofructokinase-2 (Pfk-2) is an oligomeric enzyme characterized by two kinds of interfaces: a monomer-monomer interface, critical for enzymatic activity, and a dimer-dimer interface formed upon tetramerization due to allosteric binding of MgATP. In this work, Pfk-2 was denatured by guanidine hydrochloride (GdnHCl) and the impact of ligand binding on the unfolding pathway of the dimeric and the tertrameric forms of the enzyme was examined. The unligated dimeric form unfolds and dissociates from 0.15 to 0.8 M GdnHCl without the accumulation of native monomers, as indicated by circular dichroism and size exclusion chromatography measurements. However, a monomeric intermediate with an expanded volume and residual secondary structure accumulates above 0.8 M GdnHCl. The dimeric fructose-6-P-enzyme complex shows a shift in the simultaneous dissociation and unfolding process to elevated GdnHCl concentrations (from 0.8 to 1.4 M) together with the expulsion of the ligand detected by intrinsic fluorescence measurements. The unfolding pathway of the tetrameric MgATP-enzyme complex shows the accumulation of a tetrameric intermediate with altered fluorescence properties at about 0.4 M GdnHCl. Above this concentration a sharp transition from tetramers to monomers, without the accumulation of either compact dimers or monomers, was detected by light scattering measurements. Indeed, the most populated species was a partially unfolded monomer about 0.7 M GdnHCl. On the basis of these results, we suggest that the subunit contacts are critical for the maintenance of the overall structure of Pfk-2 and for the binding of ligands, explaining the reported importance of the dimeric state for enzymatic activity.

Research paper thumbnail of Phosphate modification of fructose-1,6-bisphosphate aldolase in Escherichia coli

Biochemical and Biophysical Research Communications, 1988

When E. cell carrying multicopy plasmids for fructose-l,6-P 2 aldolase or phosphoglycerate kinase... more When E. cell carrying multicopy plasmids for fructose-l,6-P 2 aldolase or phosphoglycerate kinase was grown in the presence of 32Pi, there was label at the position of cognate high level polypeptide after SDS-PAGE. As tested for aldolase, the label was resistant to acetone, RNase, and hot TCA treatments, and was also observed by immunoprecipitation, which was competed for by purified aldolase. Incorporation Of label also occurred in the presence of chloramphenicol. Immunoprecipitation revealed apparent aldolase labeling in the wild type strain as well.

Research paper thumbnail of The structure of hemopeptide 1–65 from cytochrome c

Archives of Biochemistry and Biophysics, 1972

Hemopeptide l-65 was cleaved from horse heart cytochrome c by cyanogen bromide and purified by ex... more Hemopeptide l-65 was cleaved from horse heart cytochrome c by cyanogen bromide and purified by exclusion chromatography. Although the intrinsic viscosity of the hemopeptide is intermediate between that of a compact sphere and a random coil, the ultraviolet circular dichroic spectrum indicates no significant amount of secondary structure. A variety of chemical and spectral measurements indicate that two histidyl residues of the hemopeptide are coordinated with the heme iron, that the indole ring of tryptophan 59 is one-third exposed, that the heme moiety is two-thirds exposed, and that tyrosyl 48 and the third histidyl residue are completely exposed. It was concluded that the hemopeptide has a relatively open conformation quite different from its conformation in the native protein.

Research paper thumbnail of Influence of ligands on the aggregation of the normal and mutant forms of phosphofructokinase 2 of Escherichia coli

Archives of Biochemistry and Biophysics, 1988

Research paper thumbnail of Fructose bisphosphatase from Escherichia coli. Purification and characterization

Archives of Biochemistry and Biophysics, 1983

Escherichia coli fructose-l,&bisphosphatase has been purified for the first time, using a clone c... more Escherichia coli fructose-l,&bisphosphatase has been purified for the first time, using a clone containing an approximately 50-fold increased amount of the enzyme. The procedure includes chromatography in phosphocellulose followed by substrate elution and gel filtration. The enzyme has a subunit molecular weight of approximately 40,000 and in nondenaturing conditions is present in several aggregated forms in which the tetramer seems to predominate at low enzyme concentrations. Fructose bisphosphatase activity is specific for fructose 1,6-bisphosphate (K, of approximately 5 PM), shows inhibition by substrate above 0.05 mM, requires Me for catalysis, and has a maximum of activity around pH 7.5. The enzyme is susceptible to strong inhibition by AMP (50% inhibition around 15 PM). Phosphoenolpyruvate is a moderate inhibitor but was able to block the inhibition by AMP and may play an important role in the regulation of fructose bisphosphatase activity in wivo. Fructose 2,6-bisphosphate did not affect the rate of reaction. Fructose bisphosphatase (EC 3.1.3.11, D-fructose-1,6-bisphosphate l-phosphohydrolase) catalyzes an essential reaction of the gluconeogenic pathway and plays an important role in its regulation (1). The enzyme from several sources has been purified and characterized, but it is mainly the enzyme from mammalian tissues that has been extensively studied and whose structure and mechanism of action are relatively well known (2). The physiological role of the enzyme in Escherichia coli was shown through the use of mutants (3, 4), but the last report about the enzyme from

Research paper thumbnail of Regulatory network of the allosteric ATP inhibition of E. coli phosphofructokinase-2 studied by hybrid dimers

Biochimie, 2016

We have proposed an allosteric ATP inhibition mechanism of Pfk-2 determining the structure of dif... more We have proposed an allosteric ATP inhibition mechanism of Pfk-2 determining the structure of different forms of the enzyme together with a kinetic enzyme analysis. Here we complement the mechanism by using hybrid oligomers of the homodimeric enzyme to get insights about the allosteric communication pathways between the same sites or different ones located in different subunits. Kinetic analysis of the hybrid enzymes indicate that homotropic interactions between allosteric sites for ATP or between substrate sites for fructose-6-P have a minor effect on the enzymatic inhibition induced by ATP. In fact, the sigmoid response for fructose-6-P observed at elevated ATP concentrations can be eliminated even though the enzymatic inhibition is still operative. Nevertheless, leverage coupling analysis supports heterotropic interactions between the allosteric ATP and fructose-6-P binding occurring between and within each subunit.

Research paper thumbnail of Effect of ATP on phosphofructokinase-2 from Escherichia coli. A mutant enzyme altered in the allosteric site for MgATP

Journal of Biological Chemistry, Sep 1, 1985

The activity of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2* wa... more The activity of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2* was measured over a wide range of Mg2' and ATP concentrations. MgATP2-inhibited only the Pfk-2 enzyme, with a degree of cooperativity of 1.5. This inhibition was relieved upon increasing the fructose-6-P concentration or by lowering the pH of the reaction mixture. Other nucleotides used as phosphate donors instead of ATP did not inhibit. MgATP2-was the true substrate for both enzymes and their K,,, values for this compound were not affected by an increase of the free Mg2+ concentration. However, free Mg2' partially relieved the MgATP2-inhibition of Pfk-2 under conditions where the ATP4-concentration was negligible, without changes in the degree of cooperativity. ATP4-acted as a strong competitive inhibitor of both Pfk-2 and Pfk-2* with respect to MgATP2-with Ki values of 10 and 8 WM, respectively. ADP, AMP, and CAMP did not prevent the MgATP2-inhibition of Pfk-2. These results suggest the presence of an allosteric site for MgATP2-in Pfk-2 responsible for the MgATP2-inhibition, which is altered in Pfk-2* as a consequence of the structural mutation.

Research paper thumbnail of A phosphofructokinase mutant of Escherichia coli altered in its allosteric properties impairs gluconeogenic growth

PubMed, Dec 1, 1985

The regulation of metabolic fluxes is accomplished by modulation of key enzyme catalyzed reaction... more The regulation of metabolic fluxes is accomplished by modulation of key enzyme catalyzed reactions. This modulation takes place partially through the control of the catalytic activity of enzymes labelled as regulatory enzymes. The kinetic behavior of many regulatory enzymes can be explained in terms of multiple binding sites for effector molecules. Of these, the ones that play their control over catalysis by binding at an allosteric site have been considered of much importance. Nevertheless, proof that the effects observed in vitro, are in fact responsible for the physiological regulation in vivo, is scarce. In this regard, mutant enzymes altered in their allosteric properties might be useful. This will be illustrated with an enzyme considered crucial for the regulation of carbohydrate metabolism, namely phosphofructokinase. We present here the comparison of some of the kinetic and structural properties of wild type phosphofructokinase-2 of E. coli and of a mutant form which impairs gluconeogenic growth, an indication of the significance of the in vivo regulation. The main differences between the enzymes are their kinetic reaction mechanism, inhibitability by ATP, and aggregation states in the presence of substrates and effectors. So far these differences support only speculations as to the mechanism of the gluconeogenic impairment observed in strains that contain the mutant enzyme, a few of which are offered.

Research paper thumbnail of Crystal structure of the phosphofructokinase-2 from Escherichia coli in complex with fructose-6-phosphate

Research paper thumbnail of Ensemble Switching of the DNA-Binding Domain of Human FoxP1

Biophysical Journal, 2021

Rett Syndrome (RTT) is a rare monogenic progressive neurodevelopmental disorder occurring in abou... more Rett Syndrome (RTT) is a rare monogenic progressive neurodevelopmental disorder occurring in about 1 in 10,000 live female births. RTT is usually diagnosed within the first two years of life and manifested by loss of hand skills, impaired mobility and speech, and development of stereotypical hand movements. RTT is caused by mutations in methyl CpG-binding protein 2 (MeCP2). Several years ago, we studied the most common missense mutations in MBD domain of MeCP2 both in silico and in vitro to reveal their effect on protein structure, stability and binding to DNA. A range of methods including molecular dynamics and free energy perturbation (FEP) were employed and the predictions were compared with circular dichroism and gel mobility shift assay measurements. The 13 mutations investigated at the time were T158M,

Research paper thumbnail of Structural Dynamics of the Heterodimerization Between the DNA-Binding Domains from Human FoxP1 and FoP2 Transcription Factors

Biophysical Journal, 2021

Forkhead box P (FoxP) are members of the human Fox family of transcription factors (FoxA-FoxS), i... more Forkhead box P (FoxP) are members of the human Fox family of transcription factors (FoxA-FoxS), involved in diverse processes such as intellectual and immune system regulation. The four FoxP subfamily members share the unique ability to dimerize via domain swapping using their DNAbinding domain. Specifically, we study FoxP1 and FoxP2 dimerization due to its impact on various human diseases. Both proteins share 88% sequence identity but have distinct dimerization properties, with K D values ranging from mM (FoxP1) to mM (FoxP2). However, the structural and energetical properties of heterodimerization and the role of the DNA-binding domain and its DNA ligand in this association are unknown. In this context, we integrated single-molecule FRET and biophysical studies to probe the FoxP1-FoxP2 heterodimerization in vitro, emphasizing the effect that the ligand DNA has on the heterodimerization and the heterodimer's structural dynamics. Biophysical assessment of the heterodimerization indicated that the K D ascertained is similar to the FoxP1 homodimerization, and therefore dramatically different from the FoxP2 homodimerization value. Singlemolecule FRET analysis of FoxP1 homodimer and heterodimer (FoxP1:-FoxP2) showed that the heterodimer increases the local dynamics and the accumulation of an expanded intermediate, suggesting that FoxP2 presumably has evolved to heterodimerize instead of homodimerizing. Finally, DNAbinding assays indicated that monomer:DNA complex decreases the heterodimerization affinity, whereas having no substantial effect in the heterodimer's stability. These findings highlight relevant aspects of the regulatory role of DNA in the adoption of heterodimers and, therefore, in their gene-related expression.

Research paper thumbnail of New visible and selective DNA staining method in gels with tetrazolium salts

Analytical Biochemistry, 2017

DNA staining in gels has historically been carried out using silver staining and fluorescent dyes... more DNA staining in gels has historically been carried out using silver staining and fluorescent dyes like ethidium bromide and SYBR Green I (SGI). Using fluorescent dyes allows recovery of the analyte, but requires instruments such as a transilluminator or fluorimeter to visualize the DNA. Here we described a new and simple method that allows DNA visualization to the naked eye by generating a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in SGI and nitro blue tetrazolium (NBT) solution that, when exposed to sunlight, produces a purple insoluble formazan precipitate that remains in the gel after exposure to light. A calibration curve made with a DNA standard established a detection limit of approximately 60 pg/band at 500 bp. Selectivity of this assay was determined using different biomolecules, demonstrating a high selectivity for DNA. Integrity and functionality of the DNA recovered from gels was determined by enzymatic cutting with a restriction enzyme and by transforming competent cells after the different staining methods, respectively. Our method showed the best performance among the dyes employed. Based on its specificity, low cost and its adequacy for field work, this new methodology has enormous potential benefits to research and industry.

Research paper thumbnail of Crystal structure of the Phosphofructokinase-2 mutant Y23D from Escherichia coli

Research paper thumbnail of The alkaline isomerization of lysine-modified ferricytochrome c

Biochimica et Biophysica Acta (BBA) - Protein Structure, 1975

Both totally guanidinated and totally trifluoroacetylated ferricytochromes c exhibit a pH-depende... more Both totally guanidinated and totally trifluoroacetylated ferricytochromes c exhibit a pH-dependent displacement of the methionine ligand having an apparent pK within 0.5 pH units of that of the native protein. We propose that the pH dependence of this reaction reflects the concentration of hydroxide anion required to displace the methionine ligand.

Research paper thumbnail of An Alteration in Phosphofructokinase 2 of Escherichia coli which Impairs Gluconeogenic Growth and Improves Growth on Sugars

European Journal of Biochemistry, 2005

Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is ... more Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is allosteric, and a minor isoenzyme, phosphofructokinase 2. The pfkB1 mutation is known to increase the amount of phosphofructokinase 2 and allow growth on sugars of mutants lacking phosphofructokinase 1; it does not affect growth on substances such as glycerol or lactate (i.e., 'gluconeogenic growth'). However, gluconeogenic growth is markedly impaired in strains with a different allele, pfkB1*. We show here that strains with pfkB1* contain an altered form of phosphofructokinase 2, called phosphofructokinase 2*, which has been purified. Phosphofructokinase 2* is cold labile and has slightly different kinetic characteristics from phosphofructokinase 2, which include being less sensitive to inhibition by fructose 1,6-bisphosphate. The Km for fructose 6-phosphate is low (about 5 X 10(-5) M) in both phosphofructokinase 2 and phosphofructokinase 2*. However, in strains lacking phosphofructokinase 1, a high level of phosphofructokinase 2 is associated with unusually high concentrations of hexose monophosphates during growth on glucose, while a strain with phosphofructokinase 2* instead of phosphofructokinase 2 grows more rapidly on glucose and contains lower levels of hexose monophosphates. In gluconeogenic conditions, by contrast, hexose monophosphate levels are normal in phosphofructokinase 2 strains, while the impaired growth of phosphofructokinase 2* strains is associated with high levels of fructose 2,6-bisphosphate and very low levels of hexose monophosphates. These results show that phosphofructokinase 2, as studied in vitro, should no longer be regarded as a 'non-allosteric' protein, a conclusion also reached by Kotlarz and Buc on the basis of different types of experiments [Eur. J. Biochem. 117, 569-574 (1981)]. The fact that mutational alteration of phosphofructokinase 2 allows more rapid growth on glucose but severely impairs gluconeogenic growth is an indication of the significance of the regulation in vivo. The more rapid growth of the mutant on glucose might be explained on the basis of decreased sensitivity to an inhibitor (possibly, but not necessarily, fructose 1,6-bisphosphate), although other models are possible. A variety of speculations are offered as to the mechanism of gluconeogenic impairment.

Research paper thumbnail of An in vitro model showing different rates of substrate cycle for phosphofructokinases of Escherichia coli with different kinetic properties

European Journal of Biochemistry, 1991

An in vitro assay model is introduced for the coupled assay of phosphofructokinase (PFK) and fruc... more An in vitro assay model is introduced for the coupled assay of phosphofructokinase (PFK) and fructose-bisphosphatase. The model is applied to the study of three PFK of Escherichia coli: two isoenzymes, phosphofructokinase-1 (PFK-1) and phosphofructokinase-2 (PFK-2), and a mutant form of phosphofructokinase-2 (PFK-2*). Results show that for a variety of conditions the PFK-1/fructose-bisphosphatase pair gives the lowest and the PFK-2*/fructose-bisphosphatase pair the highest rates of substrate cycle, with the PFK-2/fructose-bisphosphatase pair in an intermediate position. The effects of variables such as maximum activity ratios and MgATP concentration were explored. The possible role of MgATP in decreasing the futile cycle of the PFK-2/fructose-bisphosphatase pair is described. The results are discussed in terms of possible metabolic consequences of PFK-2* and of predictions of the model to be tested in vivo.

Research paper thumbnail of Are the Aerobic and Anaerobic Phosphofructokinases of Escherichia coli Different?

European Journal of Biochemistry, 1977

Phosphofructokinase has been purified from Escherichia coli strain K-12 grown in a glucoselimited... more Phosphofructokinase has been purified from Escherichia coli strain K-12 grown in a glucoselimited chemostat, both aerobically and anaerobically. The enzymes migrated together in polyacrylamide gel electrophoresis, had the same subunit size in denaturing (dodecylsulfate) gels (M , approx. 34000) and the same kinetic characteristics as described earlier for E. coli phosphofructokinase [e.g. Blangy e f al. (1968) J. Mol. Biol. 31, 13-35]: a sigmoid curve of velocity vs. fructose 6-phosphate concentration, activation by ADP, and inhibition by phosphoenolpyruvate. Findings [e.g. Doelle (1975) Eur. J. Biochem. 50, 335-3421 of quite different enzymes in aerobic and anaerobic cells were not confirmed.

Research paper thumbnail of An examination of the involvement of proline peptide isomerization in protein folding

Journal of Molecular Biology, 1978

Research paper thumbnail of Reversible unfolding of dimeric phosphofructokinase-2 from Escherichia coli reveals a dominant role of inter-subunit contacts for stability

FEBS Letters, 2009

Escherichia coli phosphofructokinase-2 (Pfk-2) is a homodimer whose subunits consist of a large d... more Escherichia coli phosphofructokinase-2 (Pfk-2) is a homodimer whose subunits consist of a large domain and an additional b-sheet that provides the interfacial contacts between the subunits, creating a b-barrel flattened-like structure with the adjacent subunit's b-sheet. To determine how the structural organization of Pfk-2 determines its stability, the reversible unfolding of the enzyme was characterized under equilibrium conditions by enzymatic activity, circular dichroism, fluorescence and hydrodynamic measurements. Pfk-2 undergoes a cooperative unfolding/dissociation process with the accumulation of an expanded and unstructured monomeric intermediate with a marginal stability and a large solvent accessibility with respect to the native dimer.

Research paper thumbnail of The existence of heme-protein coordinate-covalent bonds in denaturing solvents

Research paper thumbnail of Unfolding Pathway of the Dimeric and Tetrameric Forms of Phosphofructokinase-2 from Escherichia coli †

Biochemistry, 2007

Escherichia coli phosphofructokinase-2 (Pfk-2) is an oligomeric enzyme characterized by two kinds... more Escherichia coli phosphofructokinase-2 (Pfk-2) is an oligomeric enzyme characterized by two kinds of interfaces: a monomer-monomer interface, critical for enzymatic activity, and a dimer-dimer interface formed upon tetramerization due to allosteric binding of MgATP. In this work, Pfk-2 was denatured by guanidine hydrochloride (GdnHCl) and the impact of ligand binding on the unfolding pathway of the dimeric and the tertrameric forms of the enzyme was examined. The unligated dimeric form unfolds and dissociates from 0.15 to 0.8 M GdnHCl without the accumulation of native monomers, as indicated by circular dichroism and size exclusion chromatography measurements. However, a monomeric intermediate with an expanded volume and residual secondary structure accumulates above 0.8 M GdnHCl. The dimeric fructose-6-P-enzyme complex shows a shift in the simultaneous dissociation and unfolding process to elevated GdnHCl concentrations (from 0.8 to 1.4 M) together with the expulsion of the ligand detected by intrinsic fluorescence measurements. The unfolding pathway of the tetrameric MgATP-enzyme complex shows the accumulation of a tetrameric intermediate with altered fluorescence properties at about 0.4 M GdnHCl. Above this concentration a sharp transition from tetramers to monomers, without the accumulation of either compact dimers or monomers, was detected by light scattering measurements. Indeed, the most populated species was a partially unfolded monomer about 0.7 M GdnHCl. On the basis of these results, we suggest that the subunit contacts are critical for the maintenance of the overall structure of Pfk-2 and for the binding of ligands, explaining the reported importance of the dimeric state for enzymatic activity.

Research paper thumbnail of Phosphate modification of fructose-1,6-bisphosphate aldolase in Escherichia coli

Biochemical and Biophysical Research Communications, 1988

When E. cell carrying multicopy plasmids for fructose-l,6-P 2 aldolase or phosphoglycerate kinase... more When E. cell carrying multicopy plasmids for fructose-l,6-P 2 aldolase or phosphoglycerate kinase was grown in the presence of 32Pi, there was label at the position of cognate high level polypeptide after SDS-PAGE. As tested for aldolase, the label was resistant to acetone, RNase, and hot TCA treatments, and was also observed by immunoprecipitation, which was competed for by purified aldolase. Incorporation Of label also occurred in the presence of chloramphenicol. Immunoprecipitation revealed apparent aldolase labeling in the wild type strain as well.

Research paper thumbnail of The structure of hemopeptide 1–65 from cytochrome c

Archives of Biochemistry and Biophysics, 1972

Hemopeptide l-65 was cleaved from horse heart cytochrome c by cyanogen bromide and purified by ex... more Hemopeptide l-65 was cleaved from horse heart cytochrome c by cyanogen bromide and purified by exclusion chromatography. Although the intrinsic viscosity of the hemopeptide is intermediate between that of a compact sphere and a random coil, the ultraviolet circular dichroic spectrum indicates no significant amount of secondary structure. A variety of chemical and spectral measurements indicate that two histidyl residues of the hemopeptide are coordinated with the heme iron, that the indole ring of tryptophan 59 is one-third exposed, that the heme moiety is two-thirds exposed, and that tyrosyl 48 and the third histidyl residue are completely exposed. It was concluded that the hemopeptide has a relatively open conformation quite different from its conformation in the native protein.

Research paper thumbnail of Influence of ligands on the aggregation of the normal and mutant forms of phosphofructokinase 2 of Escherichia coli

Archives of Biochemistry and Biophysics, 1988

Research paper thumbnail of Fructose bisphosphatase from Escherichia coli. Purification and characterization

Archives of Biochemistry and Biophysics, 1983

Escherichia coli fructose-l,&bisphosphatase has been purified for the first time, using a clone c... more Escherichia coli fructose-l,&bisphosphatase has been purified for the first time, using a clone containing an approximately 50-fold increased amount of the enzyme. The procedure includes chromatography in phosphocellulose followed by substrate elution and gel filtration. The enzyme has a subunit molecular weight of approximately 40,000 and in nondenaturing conditions is present in several aggregated forms in which the tetramer seems to predominate at low enzyme concentrations. Fructose bisphosphatase activity is specific for fructose 1,6-bisphosphate (K, of approximately 5 PM), shows inhibition by substrate above 0.05 mM, requires Me for catalysis, and has a maximum of activity around pH 7.5. The enzyme is susceptible to strong inhibition by AMP (50% inhibition around 15 PM). Phosphoenolpyruvate is a moderate inhibitor but was able to block the inhibition by AMP and may play an important role in the regulation of fructose bisphosphatase activity in wivo. Fructose 2,6-bisphosphate did not affect the rate of reaction. Fructose bisphosphatase (EC 3.1.3.11, D-fructose-1,6-bisphosphate l-phosphohydrolase) catalyzes an essential reaction of the gluconeogenic pathway and plays an important role in its regulation (1). The enzyme from several sources has been purified and characterized, but it is mainly the enzyme from mammalian tissues that has been extensively studied and whose structure and mechanism of action are relatively well known (2). The physiological role of the enzyme in Escherichia coli was shown through the use of mutants (3, 4), but the last report about the enzyme from

Research paper thumbnail of Regulatory network of the allosteric ATP inhibition of E. coli phosphofructokinase-2 studied by hybrid dimers

Biochimie, 2016

We have proposed an allosteric ATP inhibition mechanism of Pfk-2 determining the structure of dif... more We have proposed an allosteric ATP inhibition mechanism of Pfk-2 determining the structure of different forms of the enzyme together with a kinetic enzyme analysis. Here we complement the mechanism by using hybrid oligomers of the homodimeric enzyme to get insights about the allosteric communication pathways between the same sites or different ones located in different subunits. Kinetic analysis of the hybrid enzymes indicate that homotropic interactions between allosteric sites for ATP or between substrate sites for fructose-6-P have a minor effect on the enzymatic inhibition induced by ATP. In fact, the sigmoid response for fructose-6-P observed at elevated ATP concentrations can be eliminated even though the enzymatic inhibition is still operative. Nevertheless, leverage coupling analysis supports heterotropic interactions between the allosteric ATP and fructose-6-P binding occurring between and within each subunit.