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E Freire

Publications and source records attributed to E Freire.

At least 19 recordsLinked to original sources
PubMed1992

Microcalorimetric study of wheat germ agglutinin binding to N-acetylglucosamine and its oligomers.

The energetics of association of wheat germ agglutinin (WGA) with N-acetylglucosamine (GlcNAc) and its beta(1,4) oligomers have been measured using isothermal titration calorimetry. Association constants of 0.4, 5.3, 11.1, 12.3, and 19.1 mM-1 and enthalpies of binding of -6.1, -15.6, -19.4, -19.3, and -18.2 kcal mol-1 were obtained at 26 degrees C for the titration of WGA with GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5, respectively. The term T delta S was always of negative value, indicating that the binding process is enthalpically driven. Titrations of WGA performed at pH 4.5 did not differ significantly from those performed at pH 7.0, suggesting that no groups with a pKa in this range are directly involved in the binding event. Also, performing the titration in a buffer system with a higher enthalpy of protonation did not change the enthalpy of binding confirming that there is no net protonation or deprotonation when WGA binds GlcNAc residues at pH 7. A model of four independent binding sites was found to adequately describe the binding curves, except in the case of (GlcNAc)4 which exhibited positive cooperativity. The energetic values are discussed within the context of the structure of the WGA-(GlcNAc)2 complex.

Acetylglucosamine↗
PubMed1992

Temperature and guanidine hydrochloride dependence of the structural stability of ribonuclease T1.

The thermal unfolding of ribonuclease T1 has been studied by high-sensitivity differential scanning calorimetry as a function of temperature, [GuHCl], and scanning rate. The destabilizing effect of GuHCl has revealed that the kinetics of the unfolding transition become extremely slow as the transition temperature decreases. At pH 5.3 and zero GuHCl, the unfolding transition is centered at 59.1 degrees C; upon increasing the GuHCl concentration, the transition occurs at lower temperatures and exhibits progressively slower kinetics; so, for example, at 3 M GuHCl, the transition temperature is 40.6 degrees C and is characterized by a time constant close to 10 min. Under all conditions studied (pH 5.3, pH 7.0, [GuHCl] < 3 M), the transition is thermodynamically reversible. The slow kinetics of the transition induce significant distortions in the shape of the transition profiles that can be mistakenly interpreted as deviations from a two-state mechanism. Determination of the thermodynamic parameters from the calorimetric data has required the development of an analytical formalism that explicitly includes the thermodynamics as well as the kinetics of the transition. Using this formalism, it is shown that a two-state slow-kinetics model is capable of accurately describing the structural stability of ribonuclease T1 as a function of temperature, GuHCl concentration, and scanning rate. Multidimensional analysis of the calorimetric data has been used to estimate the intrinsic thermodynamic parameters for protein stability, the interaction parameters with GuHCl, and the time constant for the unfolding transition and its temperature dependence.

Aspergillus↗
PubMed1992

On the origin of the enthalpy and entropy convergence temperatures in protein folding.

Temperature dependence of the thermodynamics of folding/unfolding for cytochrome c has been determined as a function of moderate [0-10% (vol/vol)] concentrations of methanol. Heat capacity change (delta Cp) for unfolding decreases with increased concentrations of methanol, consistent with a higher solvent hydrophobicity. For a given transition temperature, this effect results in higher experimental enthalpy (delta H) and entropy (delta S) changes with increased methanol concentrations. When the enthalpy or entropy data sets obtained at different methanol concentrations are plotted as a function of temperature, they are seen to converge and assume common values around 100 degrees C for delta H and 112 degrees C for delta S. These convergence temperatures are similar to those obtained for different proteins in aqueous solution when delta H and delta S are normalized with respect to number of residues. It has been previously hypothesized that these convergence temperatures correspond to the temperatures at which the hydrophobic contributions to delta H and delta S are zero; the results presented here agree with this viewpoint.

Calorimetry, Differential Scanning↗
PubMed1992

Molecular basis of co-operativity in protein folding. III. Structural identification of cooperative folding units and folding intermediates.

The hierarchical partition function formalism for protein folding developed earlier has been extended through the use of three-dimensional polar and apolar contact plots. For each amino acid residue in the protein, these plots indicate the apolar and polar surfaces that are buried from the solvent, the identity of all amino acid residues that contribute to this shielding, and the magnitude of their contributions. These contact plots are then used to examine the distribution of the free energy of stabilization throughout the protein molecule. Analysis of these data allows identification of co-operative folding units and their hierarchical levels, and the identification of partially folded intermediates with a significant probability of being populated. The overall folding/unfolding thermodynamics of 12 globular proteins, for which crystallographic and experimental thermodynamics are available, is predicted within error. An energetic classification of partially folded intermediates is presented and the results compared to those cases for which structural and thermodynamic experimental information is available. Four different types of partially folded states and their structural energies are considered. (1) Local intermediates, in which only a local region of the protein loses secondary and tertiary interactions, while the rest of the protein remains intact. (2) Global intermediates, corresponding to the standard molten globule definition, in which significant secondary structure is maintained but native-like tertiary structure contacts are disrupted. (3) Extended intermediates characterized by the existence of secondary structure elements (e.g. alpha-helices) exposed to solvent. (4) Folding intermediates in proteins with two structural domains. The structure and energetics of folding intermediates of apo-myoglobin, alpha-lactalbumin, phosphoglycerate kinase and arabinose-binding protein are considered in detail.

Crystallography↗
PubMed1992

Fuc-GM1 ganglioside mimics the receptor function of GM1 for cholera toxin.

The ability of Fuc-GM1 ganglioside to mimic the receptor function of GM1 for cholera toxin (CT) has been investigated. For this purpose, rat glioma C6 cultured cells were enriched with Fuc-GM1 and the responsiveness to CT was compared with that of cells enriched with GM1 ganglioside. Fuc-GM1 was taken up by cells as rapidly and to the same extent as GM1. When comparable amounts of ganglioside were associated, the cells enriched with Fuc-GM1 bound the same amount of 125I-CT as did cells enriched with GM1. Under conditions in which GM1- and Fuc-GM1-enriched cells bound comparable amounts of CT, the Fuc-GM1-treated cells accumulated virtually the same amount of cyclic AMP as did GM1-treated cells, and activation of adenylate cyclase was also similar. The lag time preceding the CT-induced cAMP accumulation was the same in Fuc-GM1- and GM1-enriched cells. High-sensitivity isothermal titration calorimetry (ITC) experiments showed that the association constants of CT with Fuc-GM1 or GM1 ganglioside were comparable (4 x 10(7) M-1 and 1.9 x 10(7) M-1, respectively, at 25 degrees C). Also, the association constants of the B-subunit pentamer with Fuc-GM1 or GM1 ganglioside were comparable (about 3 x 10(7) M-1 and 7 x 10(7) M-1, respectively, at 25 degrees C).

Adenylyl Cyclases↗
PubMed1992

The molecular basis of cooperativity in protein folding. Thermodynamic dissection of interdomain interactions in phosphoglycerate kinase.

In the presence of guanidine hydrochloride, phosphoglycerate kinase from yeast can be reversibly denatured by either heating or cooling the protein solution above or below room temperature [Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278]. The heat denaturation of PGK is characterized by the presence of a single peak in the excess heat capacity function obtained by differential scanning calorimetry. The transition curve approaches the two-state mechanism, indicating that the two domains of the molecule display strong cooperative interactions and that partially folded intermediates are not largely populated during the transition. On the contrary, the cold denaturation is characterized by the presence of two peaks in the heat capacity function. Analysis of the data indicates that at low temperatures the two domains behave independently of each other. The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains. These interactions involve primarily the apposition of two hydrophobic surfaces of approximately 480 A2 and nine hydrogen bonds. This information, in conjunction with experimental thermodynamic values for hydrophobic, hydrogen bonding interactions and statistical thermodynamic analysis, has been used to quantitatively account for the folding/unfolding behavior of PGK. It is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration.

Calorimetry, Differential Scanning↗
PubMed1992

Two-dimensional differential scanning calorimetry: simultaneous resolution of intrinsic protein structural energetics and ligand binding interactions by global linkage analysis.

A general theoretical development for the design and analysis of two-dimensional thermal stability surfaces of proteins is presented. The surfaces are generated from multiple excess heat capacity profiles ( vs T) obtained at varying concentrations of an interacting ligand. The energetics of both the intrinsic protein stability and the protein-ligand interaction are simultaneously resolved by employing statistical thermodynamic models in global linkage analysis. This formalism allows resolution of the intrinsic protein folding-unfolding parameters (enthalpy, entropy, and heat capacity changes) as well as the ligand interaction parameters (binding stoichiometry, enthalpy, entropy, and heat capacity changes). The theory has been applied to the case of ribonuclease A and its interaction with cytidine-2'-monophosphate. The accuracy of the thermodynamic parameters obtained by this approach compares within error with those parameters that can be obtained by direct measurements.

Binding Sites↗
PubMed1991

Molecular basis of co-operativity in protein folding.

The folding/unfolding transition of proteins is a highly co-operative process characterized by the presence of very few or no thermodynamically stable partially folded intermediate states. The purpose of this paper is to present a thermodynamic formalism aimed at describing quantitatively the co-operative folding behavior of proteins. In order to account for this behavior, a hierarchical algorithm aimed at evaluating the folding/unfolding partition function has been developed. This formalism defines the partition function in terms of multiple levels of interacting co-operative folding units. A co-operative folding unit is defined as a protein structural element that exhibits two-state folding/unfolding behavior. At the most fundamental level are those structural elements that behave co-operatively as a result of purely local interactions. Higher-order co-operative folding units are formed through interactions between different structural elements. The hierarchical formalism utilizes the crystallographic structure of the protein as a template to generate partially folded conformations defined in terms of co-operative folding units. The Gibbs free energy of those states and their corresponding statistical weights are then computed using experimental energetic parameters determined calorimetrically. This formalism has been applied to the case of myoglobin. It is shown that the hierarchical partition function correctly predicts the presence, energetics and co-operativity of the heat and cold denaturation transitions. The major contribution to the co-operative folding behavior arises from the solvent exposure of non-polar residues located in regions complementary to those that have undergone unfolding. This entropically uncompensated and energetically unfavorable solvent exposure characterizes all partially folded states but not the unfolded state, thus minimizing the population of partially folded intermediates throughout the folding/unfolding transition.

Animals↗
PubMed1991

Calorimetric determination of the energetics of the molten globule intermediate in protein folding: apo-alpha-lactalbumin.

High-sensitivity differential scanning calorimetry has been used to characterize the energetics of the molten globule state of apo-alpha-lactalbumin. This characterization has been possible by performing temperature scans at different guanidine hydrochloride (GuHCl) concentrations in order to experimentally define the temperature-GuHCl stability surface of the protein. Multidimensional analysis of the heat capacity surface has allowed simultaneous resolution of the energetics of the unfolded and molten globule states. These experiments indicate that the intrinsic enthalpy difference (i.e., excluding additional contributions such as those arising from differential GuHCl binding) between the unfolded and native states is 31.8 kcal/mol at 25 degrees C whereas that of the molten globule and native states is only 7.7 kcal/mol. At the same temperature, the entropy changes are 99.2 and 23.7 cal/K.mol and the heat capacity changes are 1821 and 326 cal/K.mol, respectively. Analysis of the thermodynamic data indicates that in passing from the native to the molten globule state only approximately 19% of the hydrogen bonds are broken. In addition, the magnitude of delta Cp for the molten globule suggests that water does not largely penetrate into the interior of the molten globule, implying that significant hydrophobic interactions are still present in this state. These parameters provide precise energetic constraints to the allowed structural conformations of the molten globule.

Apoproteins↗
PubMed1991

Sugar transport by the bacterial phosphotransferase system. Structural and thermodynamic domains of enzyme I of Salmonella typhimurium.

Enzyme I, the first in the sequence of phosphocarrier proteins of the bacterial phosphoenolpyruvate:glycose phosphotransferase system, is a potential critical point for regulating sugar uptake. The thermal stability of Enzyme I was studied by high sensitivity differential scanning calorimetry. At pH 7.5, thermal unfolding of the protein exhibits two peaks with maxima (Tm) at 47.6 and 55.1 degrees C, indicating that the protein comprises two cooperative unfolding structures. Interaction between the two domains is markedly dependent on pH within the range 6.5-8.5. At pH 7.5, catalytic activity was unaffected by heating through the first transition but was lost by heating through the second. Cleavage of Enzyme I (63.5 kDa) by trypsin, chymotrypsin, or Staphylococcus aureus V8 protease yields a 30-kDa fragment, EI-N, containing the NH2 terminus and the active site, His-189. Protease and differential scanning calorimetry experiments show that EI-N is the structural domain corresponding to the cooperative region in the intact enzyme that unfolds at the higher Tm. EI-N catalyzes one activity of Enzyme I; it accepts a phosphoryl group from phosphohistidine-containing phosphocarrier protein but cannot be phosphorylated by phospho-Enzyme I or phosphoenolpyruvate. The phosphoryl transfer between EI-N and the histidine-containing phosphocarrier protein is reversible. Portions of the Salmonella typhimurium ptsI DNA sequence are known; the complete sequence is presented here and compared to Escherichia coli ptsI.

Base Sequence↗
PubMed1991

Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase.

The kinetic and thermodynamic parameters associated with the enzymatic reaction of yeast cytochrome c oxidase with its biological substrate, ferrocytochrome c, have been measured by using a titration microcalorimeter to monitor directly the rate of heat production or absorption as a function of time. This technique has allowed determination of both the energetics and the kinetics of the reaction under a variety of conditions within a single experiment. Experiments performed in buffer systems of varying ionization enthalpies allow determination of the net number of protons absorbed or released during the course of the reaction. For cytochrome c oxidase the intrinsic enthalpy of reaction was determined to be -16.5 kcal/mol with one (0.96) proton consumed for each ferrocytochrome c molecule oxidized. Activity measurements at salt concentrations ranging from 0 to 200 mM KCl in the presence of 10 mM potassium phosphate, pH 7.40, and 0.5 mM EDTA display a biphasic dependence of the electron transferase activity upon ionic strength with a peak activity observed near 50 mM KCl. The ionic strength dependence was similar for both detergent-solubilized and membrane-reconstituted cytochrome c oxidase. Despite the large ionic strength dependence of the kinetic parameters, the enthalpy measured for the reaction was found to be independent of ionic strength. Additional experiments involving direct transfer of the enzyme from low to high salt conditions produced negligible enthalpy changes that remained constant within experimental error throughout the salt concentrations studied (0-200 mM KCl). These results indicate that the salt effect on the enzyme activity is of entropic origin and further suggest the absence of a major conformational change in the enzyme due to changes in ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1991

Thermodynamic identification of stable folding intermediates in the B-subunit of cholera toxin.

The structural stability and domain structure of the pentameric B-subunit of cholera toxin have been measured as a function of different perturbants in order to assess the magnitude of the interactions within the B-subunits. For these studies, temperature, guanidine hydrochloride (GuHCl), and pH were used as perturbants, and the effects were measured by high-sensitivity differential scanning calorimetry, isothermal reaction calorimetry, fluorescence spectroscopy, and partial protease digestion. At pH 7.5 and in the absence of any additional perturbants, the thermal unfolding of the B-subunit pentamer is characterized by a single peak in the heat capacity function centered at 77 degrees C and characterized by a delta Hcal of 328 kcal/mol of B-subunit pentamer and delta Hvh/delta Hcal of 0.3. Lowering the pH down to 4 or adding GuHCl up to 2 M results in a decrease of the calorimetric enthalpy with no significant effect on the van't Hoff enthalpy. The transition enthalpy decreases in a sigmoidal fashion with pH, with an inflection point centered at pH 5.3. Isothermal titration calorimetric studies as a function of pH also report a transition centered at pH 5.3 and characterized by an enthalpy change of 27 kcal/mol of B-subunit pentamer at 27 degrees C. Below this pH, the enthalpy change for the unfolding transition is reduced to approximately 100 kcal/mol of B-subunit pentamer. Similar behavior is obtained with GuHCl. In this case, a first transition is observed at 0.5 M GuHCl and a second one at 3 M GuHCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1991

Calorimetric determination of cooperative interactions in high affinity binding processes.

It is demonstrated that isothermal titration calorimetry can be used to determine cooperative interaction energetics even for extremely tight binding processes in which the binding affinity constants are beyond the limits of experimental determination. The approach is based on the capability of calorimetry to measure the apparent binding enthalpy at any degree of ligand saturation. When calorimetric measurements are performed under conditions of total association at partial saturation, the dependence of the apparent binding enthalpy on the degree of saturation is a function only of the cooperative binding interactions. The method developed in this paper allows an independent estimation of cooperative energetic parameters without the need to simultaneously estimate or precisely know the value of the association constants. Since total ligand association at partial saturation is achieved only at macromolecular concentrations much larger than the dissociation constants, the method is especially suited for high and very high affinity processes. Biological associations in this category include fundamental cellular processes like cell surface receptor binding or protein-DNA interactions.

Binding Sites↗
PubMed1991

Multifrequency calorimetry of the folding/unfolding transition of cytochrome c.

The folding-unfolding transition of Fe(III) cytochrome c has been studied with the new technique of multifrequency calorimetry. Multifrequency calorimetry is aimed at measuring directly the dynamics of the energetic events that take place during a thermally induced transition by measuring the frequency dispersion of the heat capacity. This is done by modulating the folding/unfolding equilibrium using a variable frequency, small oscillatory temperature perturbation (approximately 0.05-0.1 degrees C) centered at the equilibrium temperature of the system. Fe(III) cytochrome c at pH 4 undergoes a fully reversible folding/unfolding transition centered at 67.7 degrees C and characterized by an enthalpy change of 81 kcal/mol and heat capacity difference between unfolded and folded states of 0.9 kcal/K*mol. By measuring the temperature dependence of the frequency dispersion of the heat capacity in the frequency range of 0.1-1 Hz it has been possible to examine the time regime of the enthalpic events associated with the transition. The multifrequency calorimetry results indicate that approximately 85% of the excess heat capacity associated with the folding/unfolding transition relaxes with a single relaxation time of 326 +/- 68 ms at the midpoint of the transition region. This is the first time that the time regime in which heat is absorbed and released during protein folding/unfolding has been measured.

Animals↗
PubMed1991

[Effect of propranolol and metoprolol on cardiorespiratory and metabolic response to exercise].

PURPOSE: To study the effects of intravenous propranolol and metoprolol on cardiorespiratory and metabolic responses to exercise. METHODS: Two groups of anaesthetized dogs (sodium pentobarbital, 33 mg/kg) were studied at rest and moderate exercise (induced by electrical stimulation) before and after metoprolol (0.4 mg/kg, n = 7) or propranolol (0.2 mg/kg, n = 10). Haemodynamic, metabolic and respiratory variables were measured. RESULTS: There was a decrease in heart rate both at rest and during exercise after metoprolol and propranolol administration. The cardiac output, stroke volume and peripheral vascular resistance presented decreased responses to exercise after both drugs. O2 consumption and CO2 production were not altered significantly at rest but increased in a lesser degree during exercise. Propranolol administration reduced ventilation during rest and exercise, although the ventilatory response to exercise remained the same. Metoprolol administration did not affect ventilation at rest but reduced the ventilatory response to exercise leading to an increase of the PaCO2. CONCLUSION: Propranolol and metoprolol effects on cardiovascular and metabolic variables measured at rest and during exercise were the same. The ventilatory effect with metoprolol administration was different from the observed with propranolol.

Animals↗
PubMed1990

Linked thermal and solute perturbation analysis of cooperative domain interactions in proteins. Structural stability of diphtheria toxin.

The temperature and guanidine hydrochloride (GuHCl) dependence of the structural stability of diphtheria toxin has been investigated by high-sensitivity differential scanning calorimetry. In 50 mM phosphate buffer at pH 8.0 and in the absence of GuHCl, the thermal unfolding of diphtheria toxin is characterized by a transition temperature (Tm) of 54.9 degrees C, a calorimetric enthalpy change (delta H) of 295 kcal/mol, and a van't Hoff to calorimetric enthalpy ratio of 0.57. Increasing the GuHCl concentration lowers the transition temperature and the calorimetric enthalpy change. At the same time, the van't Hoff to calorimetric enthalpy ratio increases until it reaches a value of 1 at 0.3 M GuHCl and remains constant thereafter. At low GuHCl concentrations (0-0.3 M), the thermal unfolding of diphtheria toxin is characterized by the presence of two transitions corresponding to the A and B domains of the protein. At higher GuHCl concentrations (0.3-1 M), the A domain is unfolded at all temperatures, and only one transition corresponding to the B domain is observed. Under these conditions, the most stable protein conformation at low temperatures is a partially folded state in which the A domain is unfolded and the B domain folded. A general model that explicitly considers the energetics of domain interactions has been developed in order to account for the stability and cooperative behavior of diphtheria toxin. It is shown that this cooperative domain interaction model correctly accounts for the temperature location as well as the shape and area of the calorimetric curves. Under physiological conditions, domain-domain interactions account for most of the structural stability of the A domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗