ATP-induced tetramerization and cooperativity in hemoglobin of lower vertebrates

The importance of intraerythrocytic organic phosphates in the allosteric control of oxygen binding to vertebrate hemoglobin (Hb) is well recognized and is correlated with conformational changes of the tetramer, ATP is a major allosteric effector of snake Hbt since the absence of this nucleotide abolishes the Hb cooperativity. This effect may be related to the molecular weight of about 32,000 for this Hb, which is compatible with the dimeric form. ATP induces a pH-dependent tetramerization of deoxyHb that leads to the recovery of cooperativity, This phenomenon may be partially explained by two amino acid replacements in the beta chains (CD2 Glu-43 --> Thr and G3 Glu-101 --> Val), which result in the loss of two negative charges at the alpha(1)beta(2) interface and favors the dissociation into dimers, The ATP-dependent dimer <-> tetramer may be physiologically important among ancient animal groups that have similar mutations and display variations in blood pH that are governed by these animals' metabolic state. The enormous loss of free energy of association that accompanies Hb oxygenation, and which is also observed at a much lower intensity in higher vertebrate Hbs, must be taken into consideration in allosteric models. We propose that the transition from a myoglobin-like protein to an allosteric one may be of evolutionary significance.27431196119

Modeling Drying Isotherms Using a Structure Transition Model

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Drying introduces structural changes in the target material that modify its interaction with water. In this work, we developed a model based on star fruit drying that considered two forms of interaction with water. This model provided a very good fit to the experimental data and was applicable to drying of other products such as apple, barley, and coffee. This model yielded better fits for data reported in the literature than other models. These findings suggest that the model is applicable to a wide range of systems.31910081019Fundacao de Amparo a Pesquisa do Estado da Bahia (FAPESB)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Pressure-induced fusogenic conformation of vesicular stomatitis virus glycoprotein

Vesicular stomatitis virus (VSV) is composed of a ribonucleoprotein core surrounded by a lipid envelope presenting an integral glycoprotein (G). The homotrimeric VSV G protein exhibits a membrane fusion activity that can be elicited by low pH. The fusion event is crucial to entry into the cell and disassembly followed by viral replication. To understand the conformational changes involved in this process, the effects of high hydrostatic pressure and urea on VSV particles and isolated G protein were investigated. With pressures up to 3.0 kbar VSV particles were converted into the fusogenic conformation, as measured by a fusion assay and by the binding of bis-ANS. The magnitude of the changes was similar to that promoted by lowering the pH. To further understand the relationship between stability and conversion into the fusion-active states, the stability of the G protein was tested against urea and high pressure. High urea produced a large red shift in the tryptophan fluorescence of G protein whereas pressure promoted a smaller change. Pressure induced equal fluorescence changes in isolated G protein and virions, indicating that virus inactivation induced by pressure is due to changes in the G protein. Fluorescence microscopy showed that pressurized particles were capable of fusing with the cell membrane without causing infection. We propose that pressure elicits a conformational change in the G protein, which maintains the fusion properties but suppresses the entry of the virus by endocytosis. Binding of bis-ANS indicates the presence of hydrophobic cavities in the G protein. Pressure also caused an increase in light scattering of VSV G protein, reinforcing the hypothesis that high pressure elicits the fusogenic activity of VSV G protein. This "fusion-intermediate state" induced by pressure has minor changes in secondary structure and is likely the cause of nonproductive infections.42185540554

PH dependence of the dissociation of multimeric hemoglobin probed by high hydrostatic pressure

We investigated the thermodynamic features of the classic alkaline dissociation of multimeric hemoglobin (3.1 MDa) from Glossoscolex paulistus (Annelidea) using high hydrostatic pressure. Light scattering measurements up to microscopic thermodynamic equilibrium indicated a high pH dependency of dissociation and association. Electron microscopy and gel filtration corroborated these findings. The volume change of dissociation decreased in absolute values from -48.0 mL/mol of subunit at pH 6.0 to -19.2 mL/mol at pH 9.0, suggesting a lack of protein interactions under alkaline conditions. Concomitantly, an increase in pH reduced the Gibbs free energy of dissociation from 37.7 to 27.5 kJ/mol of subunit. The stoichiometry of proton release calculated from the pressure-induced dissociation curves was +0.602 mol of H+/mol of subunit. These results provide a direct quantification of proton participation in stabilizing the aggregated state of the hemoglobin, and contribute to our understanding of protein-protein interactions and of the surrounding conditions that modulate the process of aggregation. (c) 2006 Elsevier B.V. All rights reserved.1254170034134

Applying structural transition theory to describe enzyme kinetics in heterogeneous systems

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Enzyme action was investigated by assuming the occurrence of different states of enzyme-substrate affinities. These states were considered to involve enzyme species with distinct abilities to form reaction product. The results obtained showed strong agreement with the experimental data for the action of peroxidase. This approach provides a powerful tool for predicting the kinetic behavior of other enzymatic processes in conditions not described before. An additional feature of this approach is the ability to characterize processes at any enzyme-substrate concentration ratio, including high enzyme-substrate ratios and enzyme inhibition by substrate or product. This proposal can also be used in systems with heterogeneity concerning the investigated enzyme.52614971513Fundacao de Amparo a Pesquisa do Estado da Bahia (FAPESB)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Substrate and enzyme concentration dependence of the Henri-Michaelis-Menten model probed by numerical simulation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)The use of the classic Henry-Michaelis-Menten (HMM) model (or simply, Michaelis-Menten model) to study the substrate and enzyme concentration dependence of enzyme catalysis is a very important step in understanding many biochemical processes, including microbial growth. Although the HMM model has been extensively studied, the conditions in which the substrate concentration is not in excess have still not been adequately defined mathematically. This lack of definition occurs despite at the cellular and molecular levels most systems generally do not operate in a state of substrate excess. In the present work, we describe an approach for studying enzyme reactions in which substrate concentrations are not in excess. Our results show that the use of extent of reactions and numerical simulation of the velocities of reaction provides an important advance in this field and furnishes results not obtained in previous studies involving these aspects. This approach, in association with knowledge of the rate constants, provides a direct and easy means of examining the single substrate-enzyme profile during product formation at any enzyme-substrate ratio. This approach is more direct than previous models that required the use of empirical equations with arbitrary constants.511144152Fundacao de Amparo a Pesquisa do Estado da Bahia (FAPESB)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Tendency for oxidation of annelid hemoglobin at alkaline pH and dissociated states probed by redox titration

The redox titration of extracellular hemoglobin of Glossoscolex paulistus (Annelidea) was investigated in different pH conditions and after dissociation induced by pressure. Oxidation increased with increasing PH, as shown by the reduced amount of ferricyanide necessary for the oxidation of hemoglobin. This behavior was the opposite of that of vertebrate hemoglobins. The potential of half oxidation (E 1/2) changed from - 65.3 to + 146.8 mV when the pH increased from 4.50 to 8.75. The functional properties indicated a reduction in the log P-50 from 1.28 to 0.28 in this pH range. The dissociation at alkaline pH or induced by high pressure, confirmed by HPLC gel filtration, suggested that disassembly of the hemoglobin could be involved in the increased potential for oxidation. These results suggest that the high stability and prolonged lifetime common to invertebrate hemoglobins is related to their low tendency to oxidize at acidic pH, in contrast to vertebrate hemoglobins. (c) 2005 Elsevier Inc. All rights reserved.141449850

Entropy and Volume Change of Dissociation in Tobacco Mosaic Virus Probed by High Pressure

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Virus dissociation and inactivation by high pressure have been extensively studied in recent decades. Pressure-induced dissociation of viral particles involves a reduction in the Gibbs free energy of dissociation and a negative change in volume. In this work, we investigated the combined effect of high pressure and temperature on the dissociation of tobacco mosaic virus (TMV). We assumed the presence of two states of TMV with different tendencies to dissociate. Thus one form presents a low tendency (L) and the other a high tendency (H) to dissociate. Based on the model described here, the L H transition was favored by an increase in pressure and a decrease in temperature. The volume change of dissociation was pressure- and temperature-dependent, with a highly negative value of -80 mL/mol being recorded at 0 degrees C and atmospheric pressure. The entropy and enthalpy of dissociation were very temperature- and pressure-dependent, with values of entropy of 450 to -1300 kJ/mol and values of enthalpy of 5.5 X 10(4) to 2.4 x 10(4) kJ/mol. The dissociation of TMV was enthalpy-driven at all temperatures and pressures investigated. Based on these findings, we conclude that the model presented allows accurate predictions of viral dissociation behavior in different experimental conditions.116511481714828Fundacao de Amparo a Pesquisa do Estado da Bahia (FAPESB)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Different urea stoichiometries between the dissociation and denaturation of tobacco mosaic virus as probed by hydrostatic pressure

Viruses are very efficient self-assembly structures, but little is understood about the thermodynamics governing their directed assembly. At higher levels of pressure or when pressure is combined with urea, denaturation occurs. For a better understanding of such processes, we investigated the apparent thermodynamic parameters of dissociation and denaturation by assuming a steady-state condition. These processes can be measured considering the decrease of light scattering of a viral solution due to the dissociation process, and the red shift of the fluorescence emission spectra, that occurs with the denaturation process. We determined the apparent urea stoichiometry considering the equilibrium reaction of TMV dissociation and subunit denaturation, which furnished, respectively, 1.53 and 11.1 mol of urea/mol of TMV subunit. The denaturation and dissociation conditions were arrived in a near reversible pathway, allowing the determination of thermodynamic parameters. Gel filtration HPLC, electron microscopy and circular dichroism confirmed the dissociation and denaturation processes. Based on spectroscopic results from earlier papers, the calculation of the apparent urea stoichiometry of dissociation and denaturation of several other viruses resulted in similar values, suggesting a similar virus-urea interaction among these systems. (c) 2008 Elsevier B.V. All rights reserved.134321422