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At least 1,135 records · Page 63Linked to original sources
PubMed2004

Thermodynamics of ligand binding by the yeast mRNA-capping enzyme reveals different modes of binding.

RNA-capping enzymes are involved in the synthesis of the cap structure found at the 5'-end of eukaryotic mRNAs. The present study reports a detailed study on the thermodynamic parameters involved in the interaction of an RNA-capping enzyme with its ligands. Analysis of the interaction of the Saccharomyces cerevisiae RNA-capping enzyme (Ceg1) with GTP, RNA and manganese ions revealed significant differences between the binding forces that drive the interaction of the enzyme with its RNA and GTP substrates. Our thermodynamic analyses indicate that the initial association of GTP with the Ceg1 protein is driven by a favourable enthalpy change (DeltaH=-80.9 kJ/mol), but is also clearly associated with an unfavourable entropy change (TDeltaS=-62.9 kJ/mol). However, the interaction between Ceg1 and RNA revealed a completely different mode of binding, where binding to RNA is clearly dominated by a favourable entropic effect (TDeltaS=20.5 kJ/mol), with a minor contribution from a favourable enthalpy change (DeltaH=-5.3 kJ/mol). Fluorescence spectroscopy also allowed us to evaluate the initial binding of GTP to such an enzyme, thereby separating the GTP binding step from the concomitant metal-dependent hydrolysis of GTP that results in the formation of a covalent GMP-protein intermediate. In addition to the determination of the energetics of ligand binding, our study leads to a better understanding of the molecular basis of substrate recognition by RNA-capping enzymes.

Cations↗
PubMed1984

A two-state thermodynamic and kinetic analysis of the allosteric functioning of the haemoglobin of an extreme poikilotherm.

The blood of the extreme poikilotherm Trematomus borchgrevinki contains one major haemoglobin component, which may be separated from the minor species by ion-exchange chromatography. This haemoglobin shows co-operative CO-binding isotherms at pH 8.2. An analysis of the temperature-dependence of the binding curves has allowed the thermodynamic constants associated with the two-state allosteric parameters L, KR and KT to be measured. The binding of CO at lower pH (6.2) is characterized by the maintenance of the T-state to relatively high degrees of saturation. Kinetic investigations with the use of flash photolysis of the haemoglobin-CO complex under various conditions has allowed the determination of the thermodynamic parameters association with the T-state and R-state rate constants. An amalgamation of these data has allowed the mathematical simulation of predicted time courses for CO binding to this haemoglobin, by using the two-state model, which very closely represents those obtained experimentally. The overall findings show that this haemoglobin closely follows the two-state model of co-operative interaction.

Allosteric Regulation↗
PubMed1986

Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin.

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 X 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = -34.7 kJ X mol-1, delta H = -37.9 kJ X mol-1 and delta S = -10.9 J X mol-1 X K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 X 10(5) M-1 X s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.

Acetylgalactosamine↗
PubMed1990

Thermodynamic analysis of the interaction of the antibiotic teicoplanin and its aglycone with cell-wall peptides.

The thermodynamics of the interaction of the glycopeptidic antibiotic teicoplanin and its peptidic moiety with analogues of bacterial cell-wall peptides were studied by means of calorimetric and spectrophotometric techniques. The analysis of the thermodynamic data has allowed us to evaluate the contributions of the different peptide groups to the binding process. The nature of the primary binding forces is also discussed for each interacting group, on the basis of their enthalpic and entropic contribution and in connection with the detailed structural information available for these antibiotics from n.m.r. data. Similar analyses for the case of vancomycin and ristocetin are also reported.

Amino Acid Sequence↗
PubMed1996

Effect of substituent on the thermodynamics of D-glucopyranoside binding to concanavalin A, pea (Pisum sativum) lectin and lentil (Lens culinaris) lectin.

Titration calorimetry measurements of the binding of phenyl-alpha (alpha PhOGlu), 3-methoxy (3MeOGlu), fluorodeoxy and deoxy derivatives of alpha-D-glucopyranose (Glu) to concanavalin A (conA), pea lectin and lentil lectin were performed at approx. 10 and 25 degrees C in 0.01 M dimethylglutaric acid/NaOH buffer, pH 6.9, containing 0.15 M NaCl and Mn2+ and Ca2+ ions. Apparently the 3-deoxy, 4-deoxy and 6-deoxy as well as the 4-fluorodeoxy and 6-fluorodeoxy derivatives of Glu do not bind to the lectins because no heat release was observed on the addition of aliquots of solutions of these derivatives to the lectin solutions. The binding enthalpies, delta H0b, and entropies, delta S0b, determined from the measurements were compared with the same thermodynamic binding parameters for Glu, D-mannopyranoside and methyl-alpha- D-glucopyranoside (alpha MeOGlu). The binding reactions are enthalpically driven with little change in the heat capacity on binding, and exhibit enthalpy-entropy compensation. Differences between the thermodynamic binding parameters can be rationalized in terms of the interactions apparent in the known crystal structures of the methyl-alpha-D-mannopyranoside-conA [Derewenda, Yariv, Helliwell, Kalb (Gilboa), Dodson, Papiz, Wan and Campbell (1989) EMBO J. 8, 2189-2193] and pea lectin-trimanno-pyranoside [Rini, Hardman, Einspahr, Suddath and Carber (1993) J. Biol. Chem. 268, 10126-10132] complexes. Increases in the entropy change on binding are observed for alpha MeOGlu binding to pea and lentil lectin, for alpha PhOGlu binding to conA and pea lectin, and for 3MeOGlu binding to pea lectin relative to the entropy change for Glu binding, and imply that the phenoxy and methoxy substituents provide additional hydrophobic interactions in the complex. Increases in the binding enthalpy relative to that of Glu are observed for deoxy and fluoro derivatives in the C-1 and C-2 positions and imply that these substituents weaken the interaction with the surrounding water, thereby strengthening the interaction with the binding site.

Binding Sites↗
PubMed1996

Thermodynamic studies of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast microsomes.

An approach to the quantitative spectral analysis of substrate binding and inactivation of cytochrome P-450 in microsomes is described. The method is based on the application of the principal component analysis technique on the Soret-region spectra measured at different temperatures at various concentrations of substrate. This approach allowed us to study the thermodynamic parameters of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast (Saccharomyces cerevisiae) microsomes. These parameters are discussed in comparison with the values reported earlier by Ristau et al. [(1979) Acta Biol. Med. Ger. 38, 177-185] for rabbit liver cytochrome P-450 2B4 in solution with benzphetamine as a substrate. Our analysis shows the substrate-free states of 2B4 and 3A4 to be very similar. However, substrate binding seems to perturb haem-protein interactions in 3A4 in contrast with 2B4, where the effect of substrate binding on the thermodynamic parameters of spin transitions was insignificant. The implication of the results for the mechanism of substrate-induced spin shift is discussed.

Animals↗
PubMed2001

Thermodynamic and kinetic analysis of porphyrin binding to Trichosanthes cucumerina seed lectin.

The interaction of several metallo-porphyrins with the galactose-specific lectin from Trichosanthes cucumeirna (TCSL) has been investigated. Difference absorption spectroscopy revealed that significant changes occur in the Soret band region of the porphyrins upon binding to TCSL and these changes have been monitored to obtain association constants (Ka) and stoichiometry of binding (n). The dimeric lectin binds two porphyrin molecules and the presence of the specific saccharide lactose did not affect porphyrin binding significantly, indicating that the sugar and the porphyrin bind at different sites. The Ka values obtained for the binding of different porphyrins with TCSL at 25 degrees C were in the range of 2 x 10(3)-5 x 10(5) m(-1). Association constants for meso-tetra(4-sulphonatophenyl)porphyrinato copper(II) (CuTPPS), a porphyrin bearing four negative charges and meso-tetra(4-methylpyridinium)porphyrinato copper(II) (CuTMPyP), a porphyrin with four positive charges, were determined at several temperatures; from the temperature dependence of the association constants, the thermodynamic parameters change in enthalpy (DeltaH degrees ) and change in entropy (DeltaS degrees ) associated with the binding process were estimated. The thermodynamic data indicate that porphyrin binding to TCSL is driven largely by a favourable entropic contribution; the enthalpic contribution is very small, suggesting that the binding process is governed primarily by hydrophobic forces. Stopped-flow spectroscopic measurements show that binding of CuTMPyP to TCSL takes place by a single-step process and at 20 degrees C, the association and dissociation rate constants were 1.89 x 10(4) m(-1).s(-1) and 0.29 s(-1), respectively.

Absorption↗
PubMed2001

Thermodynamics of the folding of D-glyceraldehyde-3-phosphate dehydrogenase assisted by protein disulfide isomerase studied by microcalorimetry.

Thermodynamics of the refolding of denatured D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) assisted by protein disulfide isomerase (PDI), a molecular chaperone, has been studied by isothermal microcalorimetry at different molar ratios of PDI/GAPDH and temperatures using two thermodynamic models proposed for chaperone-substrate binding and chaperone-assisted substrate folding, respectively. The binding of GAPDH folding intermediates to PDI is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong enthalpy-entropy compensation and weak temperature dependence of Gibbs free energy change. A large negative heat-capacity change of the binding, -156 kJ.mol(-1).K(-1), at all temperatures examined indicates that hydrophobic interaction is a major force for the binding. The binding stoichiometry shows one dimeric GAPDH intermediate per PDI monomer. The refolding of GAPDH assisted by PDI is a largely exothermic reaction at 15.0-25.0 degrees C. With increasing temperature from 15.0 to 37.0 degrees C, the PDI-assisted reactivation yield of denatured GAPDH upon dilution decreases. At 37.0 degrees C, the spontaneous reactivation, PDI-assisted reactivation and intrinsic molar enthalpy change during the PDI-assisted refolding of GAPDH are not detected.

Animals↗
PubMed2001

Thermodynamic analysis of the binding of glutathione to glutathione S-transferase over a range of temperatures.

The binding properties of a glutathione S-transferase (EC 2.5.1.18) from Schistosoma japonicum to substrate glutathione (GSH) has been investigated by intrinsic fluorescence and isothermal titration calorimetry (ITC) at pH 6.5 over a temperature range of 15-30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of GSH at pH 6.5. We have also studied the effect of pH on the thermodynamics of GSH-GST interaction. The behaviour shown at different pHs indicates that at least three groups must participate in the exchange of protons. Fluorimetric and calorimetric measurements indicate that GSH binds to two sites in the dimer of 26-kDa glutathione S-transferase from Schistosoma japonicum (SjGST). On the other hand, noncooperativity for substrate binding to SjGST was detected over a temperature range of 15-30 degrees C. Among thermodynamic parameters, whereas DeltaG degrees remains practically invariant as a function of temperature, DeltaH and DeltaS degrees both decrease with an increase in temperature. While the binding is enthalpically favorable at all temperatures studied, at temperatures below 25 degrees C, DeltaG degrees is also favoured by entropic contributions. As the temperature increases, the entropic contributions progressively decrease, attaining a value of zero at 24.3 degrees C, and then becoming unfavorable. During this transition, the enthalpic contributions become progressively favorable, resulting in an enthalpy-entropy compensation. The temperature dependence of the enthalpy change yields the heat capacity change (DeltaCp degrees ) of -0.238 +/- 0.04 kcal per K per mol of GSH bound.

Animals↗
PubMed2001

Impact of surface thermodynamics on bacterial transport.

Microbial surface thermodynamics correlated with bacterial transport in saturated porous media. The surface thermodynamics was characterized by contact-angle measurement and the wicking method, which was related to surface free energies of Lifshitz-van der Waals interaction, Lewis acid-base interaction, and electrostatic interaction between the bacteria and the medium matrix. Transport of three different strains of bacteria present at three physiological states was measured in columns of silica gel and sand from the Canadian River Alluvium (Norman, OK, USA). Microorganisms in stationary state had the highest deposit on solid matrix, compared with logarithmic and decay states. The deposition correlated with the total surface free energy (DeltaG132TOT) and the differences in DeltaG132TOT were mainly controlled by the Lewis acid-base interaction. Infrared spectroscopy showed that the increased deposition correlated with an increase in the hydrogen-bonding functional groups on the cell surfaces.

Bacterial Adhesion↗
PubMed1995

Thermodynamic parameters of frog brain kappa-opioid receptors.

The thermodynamic parameters for [3H]-ethylketocyclazocine binding in frog (Rana esculenta) brain membranes have been examined. Computer-based nonlinear regression analysis of the untransformed equilibrium displacement data showed that this ligand bound to two sites with different affinities and capacities in this tissue. KA values derived from equilibrium displacement curves have been used for calculating the changes in the standard Gibbs energy, enthalpy, and entropy during the binding process. Van't Hoff plots are bipartite, with transitions occurring at 18 degrees C for both the high- and the low-affinity sites. For the high-affinity site, the reaction appears to be associated with a decrease in enthalpy below the transition temperature and a significant gain in entropy above this temperature. The reverse appears to be true for the low-affinity site. We conclude that this profile fairly approximates the mixed agonist-antagonist nature of this ligand and surmise that thermodynamic analysis could be a very useful tool for characterization of the nature of cloned opioid receptors in vitro.

Animals↗
PubMed1996

Distinct thermodynamic parameters of serotonin 5-HT3 agonists and antagonists to displace [3H]granisetron binding.

Specific binding of [3H]granisetron was examined to serotonin 5-HT3 receptors in synaptosomal membranes of rat cerebral cortex between 1 and 37 degrees C. Displacing potencies were determined for 5-HT3 antagonists (granisetron, ondansetron, tropisetron, and d-tubocurarine) and agonists (5-hydroxytryptamine, 2-methyl-5-hydroxytryptamine, phenylbiguanide, m-chlorophenylbiguanide, and SR 57227A). Displacing potencies of the agonists decreased with decreasing temperature. In contrast, displacing potencies of all antagonists increased with decreasing temperature, whereas those of tropisetron and d-tubocurarine passed a maximum. Scatchard analysis of [3H]granisetron binding resulted in KD values lower than the IC50 values of granisetron and a decreasing number of binding sites at higher temperatures. It can be reconciled with temperature-dependent agonist and antagonist states of 5-HT3 receptors. A semiquantitative thermodynamic analysis was based on displacing potencies. The distinct patterns for the signs of entropy, enthalpy, and heat capacity changes on binding can be reconciled with ionic interactions for agonists and hydrophobic interactions for antagonists. The distinctive differences in these thermodynamic parameters exceed those for GABAA and glycine receptor-ionophore complexes.

Animals↗
PubMed2004

Thermodynamics of lattice heteropolymers.

We calculate thermodynamic quantities of hydrophobic-polar (HP) lattice proteins by means of a multicanonical chain-growth algorithm that connects the new variants of the Pruned-Enriched Rosenbluth Method and flat histogram sampling of the entire energy space. Since our method directly simulates the density of states, we obtain results for thermodynamic quantities of the system for all temperatures. In particular, this algorithm enables us to accurately simulate the usually difficult accessible low-temperature region. Therefore, it becomes possible to perform detailed analyses of the low-temperature transition between ground states and compact globules.

Algorithms↗
PubMed2004

Thermodynamics of beta-amyloid fibril formation.

Amyloid fibers are aggregates of proteins. They are built out of a peptide called beta-amyloid (Abeta) containing between 41 and 43 residues, produced by the action of an enzyme which cleaves a much larger protein known as the amyloid precursor protein (APP). X-ray diffraction experiments have shown that these fibrils are rich in beta-structures, whereas the shape of the peptide displays an alpha-helix structure within the APP in its biologically active conformation. A realistic model of fibril formation is developed based on the 17 residues Abeta12-28 amyloid peptide, which has been shown to form fibrils structurally similar to those of the whole Abeta peptide. With the help of physical arguments and in keeping with experimental findings, the Abeta12-28 monomer is assumed to be in four possible states (i.e., native helix conformation, beta-hairpin, globular low-energy state, and unfolded state). Making use of these monomeric states, oligomers (dimers, tertramers, and octamers) were constructed. With the help of short, detailed molecular dynamics calculations of the three monomers and of a variety of oligomers, energies for these structures were obtained. Making use of these results within the framework of a simple yet realistic model to describe the entropic terms associated with the variety of amyloid conformations, a phase diagram can be calculated of the whole many-body system, leading to a thermodynamical picture in overall agreement with the experimental findings. In particular, the existence of micellar metastable states seem to be a key issue to determine the thermodynamical properties of the system.

Amino Acid Sequence↗
PubMed2005

Thermodynamics of Go-type models for protein folding.

Go-type potentials, based on the inter-residue contacts present in the native structure of a protein, are frequently used to predict dynamic and structural features of the folding pathways through computer simulations. However, the mathematical form used to define the model interactions includes several arbitrary choices, whose consequences are not usually analyzed. In this work, we use a simple off-lattice protein model and a parallel tempering Monte Carlo simulation technique to carry out such analysis, centered in the thermodynamic characteristics of the folding transition. We show how the definition of a native contact has a deep impact on the presence of simple or complex transitions, with or without thermodynamic intermediates. In addition, we have checked that the width of the attractive wells has a profound effect on the free-energy barrier between the folded and unfolded states, mainly through its influence on the entropy of the denatured state.

Computer Simulation↗
PubMed2005

Thermodynamic prediction of protein neutrality.

We present a simple theory that uses thermodynamic parameters to predict the probability that a protein retains the wild-type structure after one or more random amino acid substitutions. Our theory predicts that for large numbers of substitutions the probability that a protein retains its structure will decline exponentially with the number of substitutions, with the severity of this decline determined by properties of the structure. Our theory also predicts that a protein can gain extra robustness to the first few substitutions by increasing its thermodynamic stability. We validate our theory with simulations on lattice protein models and by showing that it quantitatively predicts previously published experimental measurements on subtilisin and our own measurements on variants of TEM1 beta-lactamase. Our work unifies observations about the clustering of functional proteins in sequence space, and provides a basis for interpreting the response of proteins to substitutions in protein engineering applications.

Amino Acid Substitution↗
PubMed2002

Roles of mutation and recombination in the evolution of protein thermodynamics.

We present a comprehensive study of the evolutionary origin of the thermodynamic behavior of proteins. With the use of a simplified model, we exhaustively enumerate the space of all sequences and the space of all structures, simulate the evolutionary relationship between sequences and structures, and characterize the steady-state sequence distribution for all structures in terms of several thermodynamic variables. We assess the effects of two major forces of evolution: mutation and recombination. Three simplifications are made. First, a two-dimensional lattice model is used to represent protein sequences and structures. Second, proteins undergo neutral evolution so that the fitness landscape has a flat allowed region inside of which all sequences are equally fit. Third, we ignore otherwise important factors such as finite population size and evolutionary time. Two scenarios emerge from our study. The first occurs when evolution is dominated by mutation events. Even though the prototype sequence that is most mutationally robust is preferred by evolution, the preference is not strong enough to offset the huge size of sequence space. Most native sequences are located near the boundary of the fitness region and are marginally compatible with the native structure. The second scenario occurs when evolution is dominated by recombination events. Now evolutionary preference for prototype sequence is strong enough to overcome the size of sequence space so that most native sequences are located near the center of sequence-structure compatibility. We conclude that the relative frequency of mutation and recombination events is a major determinant of how optimal protein sequences are for their structures.

Evolution, Molecular↗
PubMed1976

Chemical reactivities of catalytic and noncatalytic zinc or cobalt atoms of horse liver alcohol dehydrogenase: differentiation by their thermodynamic and kinetic properties.

Horse liver alcohol dehydrogenase (EC 1.1.1.1) contains one catalytic and one noncatalytic pair of zinc atoms that can be replaced selectively with cobalt and/or 65zince. We have now prepared "hybird" metalloenzymes by specifically replacing one or both pairs of zinc atoms with 65zinc and/or cobalt. Their differential chemical reactivities serve to characterize the metal atoms at either site. The spectral and kinetic properties of the resultant 65zinc, cobalt, and hybrid enzymes, as well as those of their complexes with 1,10-phenanthroline, identify the metal atoms that are at the catalytic sites and differentiate them from those at the noncatalytic sites. All data are in complete agreement with the results of the x-ray crystal structure analysis. Remarkably, under the conditions used, chemical reactivity, as gauged by thermodynamic methods under equilibrium conditions, identifies the catalytic metal atoms as those which are reactive to 1,10-phenanthroline, while this reagnet does not affect the noncatalytic pair. Under dynamic conditions the kinetics of the metal-metal exchange reveals the converse to be true: the chemical reactivity of the noncatalytic atoms is much higher and, hence, they exchange more rapidly. The results are examined in terms of thermodynamic and kinetic properties of metal complex ions which serve as the basis of possible mechanisms underlying these observations.

Alcohol Oxidoreductases↗