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PubMed1995

Comparisons of the thermodynamic properties of three nickel-titanium orthodontic archwires.

The unique memory property of thermodynamic wire is only partially understood. It is believed to result from the wire's inherent capability to markedly alter its atomic bonding forces as a function of temperature. This shape recovery phenomenon may be the result of a transition in crystal structure that occurs by deformation and cooling. When the transition is reversed, by heating, the structure reverts to its original form and abrupt property changes occur. The purpose of this study was to determine the transition temperature ranges (TTR) of three commercially available thermodynamic archwires and to determine the rate of recovery of the wires when bent to a uniform shape. A jig was constructed to hold the wires and was suspended in a water bath within a plexiglass box. The temperature of the water bath was gradually increased. A program was written to acquire a single video frame from a running video tape and then allow the operator to graphically overlay the position of each wire specimen. The results indicate that the TTRs for the three commercially available thermodynamic wires are of similar magnitudes (x = 6.7 degrees C, 6.2 degrees C and 6.7 degrees C). The greatest differences were in the standard deviations (1.3 degrees C, 2.2 degrees C and 3.7 degrees C) which may be a function of manufacturing during alloying of the wire and/or its heat treatment.

Analysis of Variance↗
PubMed2000

Thermodynamic and kinetic analyses for understanding sequence-specific DNA recognition.

Thermodynamic and kinetic analyses of biomolecular interactions reveal details of the energetic and dynamic features of molecular recognition processes, and complement structural analyses of the free and complexed conformations. The recent improvements in both isothermal titration calorimetry and surface plasmon resonance sensoring provide powerful tools for analysing biomolecular interactions in thermodynamic and kinetic approaches. The thermodynamic and kinetic parameters obtained for binding between protein and DNA indicate the mechanism of specific DNA recognition, in the high-resolution structures of the protein-DNA complexes. The effects of temperature and ionic strength reflect the conformational changes of the protein and DNA molecules upon complex formation, including important contributions of water and solutes. When combined with mutational studies, the interactions can be reduced to several energetic contributions from individual contacts. These studies should be useful to determine general features of protein functions in genetic regulation.

Animals↗
PubMed2002

Thermodynamic and kinetic characterization of trihaem cytochrome c3 from Desulfuromonas acetoxidans.

Trihaem cytochrome c3 (also known as cytochrome c551.5 and cytochrome c7) is isolated from the periplasmic space of Desulfuromonas acetoxidans, a sulfur-reducing bacterium. Thermodynamic and kinetic data for the trihaem cytochrome c3 are presented and discussed in the context of the possible physiological implications of its functional properties with respect to the natural habitat of D. acetoxidans, namely as a symbiont with green sulfur bacteria working as a mini-sulfuretum. The thermodynamic properties were determined through the fit of redox titration data, followed by NMR and visible spectroscopy, to a model of four functional centres that describes the network of cooperativities between the three haems and one protolytic centre. The kinetics of trihaem cytochrome c3 reduction by sodium dithionite were studied using the stopped-flow technique and the data were fitted to a kinetic model that makes use of the thermodynamic properties to obtain the rate constants of the individual haems. This analysis indicates that the electrons enter the cytochrome mainly via haem I. The reduction potentials of the haems in this cytochrome show little variation with pH within the physiological range, and the kinetic studies show that the rates of reduction are also independent of pH in the range studied. Thus, although the trihaem cytochrome c3 is readily reduced by hydrogenases from Desulfovibrio sp. and its haem core is similar to that of the homologous tetrahaem cytochromes c3, its physico-chemical properties are quite different, which suggests that these multihaem cytochromes with similar structures perform different functions.

Cytochrome c Group↗
PubMed2004

Thermodynamic stability and kinetic foldability of a lattice protein model.

By using serial mutations, i.e., a residue replaced by 19 kinds of naturally occurring residues, the stability of native conformation and folding behavior of mutated sequences are studied. The 3 x 3 x 3 lattice protein model with two kinds of interaction potentials between the residues, namely the original Miyazawa and Jernigan (MJ) potentials and the modified MJ potentials (MMJ), is used. Effects of various sites in the mutated sequences on the stability and foldability are characterized through the Z-score and the folding time. It is found that the sites can be divided into three types, namely the hydrophobic-type (H-type), the hydrophilic-type (P-type) and the neutral-type (N-type). These three types of sites relate to the hydrophobic core, the hydrophilic surface and the parts between them. The stability of the native conformation for the serial mutated sequences increases (or decreases) as the increasing in the hydrophobicity of the mutated residues for the H-type sites (or the P-type sites), while varies randomly for the N-type sites. However, the foldability of the mutated sequences is not always consistent with the thermodynamic stability, and their relationship depends on the site types. Since the hydrophobic tendency of the MJ potentials is strong, the ratio between the number of the H-type sites and the number of the P-type sites is found to be 1:2. Differently, for the MJJ potentials it is found that such a ratio is about 1:1 which is relevant to that of real proteins. This suggests that the modification of the MJ potentials is rational in the aspect of thermodynamic stability. The folding of model proteins with the MMJ potentials is fast. However, the relationship between the foldability and the thermodynamic stability of the mutated sequences is complex.

Hydrophobic and Hydrophilic Interactions↗
PubMed2006

Disparate thermodynamics governing T cell receptor-MHC-I interactions implicate extrinsic factors in guiding MHC restriction.

The underlying basis of major histocompatibility complex (MHC) restriction is unclear. Nevertheless, current data suggest that a common thermodynamic signature dictates alphabeta T cell receptor (TcR) ligation. To evaluate whether this thermodynamic signature defines MHC restriction, we have examined the thermodynamic basis of a highly characterized immunodominant TcR interacting with its cognate peptide-MHC-I ligand. Surprisingly, we observed this interaction to be governed by favorable enthalpic and entropic forces, which is in contrast to the prevailing generality, namely, enthalpically driven interactions combined with markedly unfavorable entropic forces. We conclude that extrinsic molecular factors, such as coreceptor ligation, conformational adjustments involved in TcR signaling, or constraints dictated by higher-order arrangement of ligated TcRs, might play a greater role in guiding MHC restriction than appreciated previously.

Amino Acid Sequence↗
PubMed2002

The evolution of multicomponent systems at high pressures: VI. The thermodynamic stability of the hydrogen-carbon system: the genesis of hydrocarbons and the origin of petroleum.

The spontaneous genesis of hydrocarbons that comprise natural petroleum have been analyzed by chemical thermodynamic-stability theory. The constraints imposed on chemical evolution by the second law of thermodynamics are briefly reviewed, and the effective prohibition of transformation, in the regime of temperatures and pressures characteristic of the near-surface crust of the Earth, of biological molecules into hydrocarbon molecules heavier than methane is recognized. For the theoretical analysis of this phenomenon, a general, first-principles equation of state has been developed by extending scaled particle theory and by using the technique of the factored partition function of the simplified perturbed hard-chain theory. The chemical potentials and the respective thermodynamic Affinity have been calculated for typical components of the H-C system over a range of pressures between 1 and 100 kbar (1 kbar = 100 MPa) and at temperatures consistent with those of the depths of the Earth at such pressures. The theoretical analyses establish that the normal alkanes, the homologous hydrocarbon group of lowest chemical potential, evolve only at pressures greater than approximately 30 kbar, excepting only the lightest, methane. The pressure of 30 kbar corresponds to depths of approximately 100 km. For experimental verification of the predictions of the theoretical analysis, a special high-pressure apparatus has been designed that permits investigations at pressures to 50 kbar and temperatures to 1,500 degrees C and also allows rapid cooling while maintaining high pressures. The high-pressure genesis of petroleum hydrocarbons has been demonstrated using only the reagents solid iron oxide, FeO, and marble, CaCO3, 99.9% pure and wet with triple-distilled water.

Carbon↗
PubMed2001

Thermodynamics "beyond" local equilibrium.

Nonequilibrium thermodynamics has shown its applicability in a wide variety of different situations pertaining to fields such as physics, chemistry, biology, and engineering. As successful as it is, however, its current formulation considers only systems close to equilibrium, those satisfying the so-called local equilibrium hypothesis. Here we show that diffusion processes that occur far away from equilibrium can be viewed as at local equilibrium in a space that includes all the relevant variables in addition to the spatial coordinate. In this way, nonequilibrium thermodynamics can be used and the difficulties and ambiguities associated with the lack of a thermodynamic description disappear. We analyze explicitly the inertial effects in diffusion and outline how the main ideas can be applied to other situations.

Biology↗
PubMed2001

Hydration of the peptide backbone largely defines the thermodynamic propensity scale of residues at the C' position of the C-capping box of alpha-helices.

The C' position of the C-capping box is the second residue outside of the helix. Statistical analysis of residue distribution at the C' position in the alpha-helices' C-capping box showed that different amino acid residues occur with different probabilities, with the strongest preference being for glycine. To understand the physico-chemical basis for this preference, we studied the effects that 17 amino acid substitutions at the C' position in an alpha-helix of ubiquitin have on the stability of this protein. We determined the following rank order of amino acid residues at the C' position with respect to their effect on the stability: Gly>His>Asn>Arg>Lys>Gln>Ala>Phe>Met>Ser>Asp>Glu>Trp>Thr>Pro>Ile>Val. The effect of the amino acid substitutions on the structure also was evaluated by comparing the (1)H-(15)N heteronuclear sequential quantum correlation spectra and showed no significant changes in the structures of the most stable (Gly) and the least stable (Val) variants. The obtained changes in stability highly correlate (r = 0.85) with the statistical distribution of the residues at the C' position indicating that the measured thermodynamic propensities are unbiased by secondary interactions. We also found that the measured thermodynamic propensities correlate well with the amide hydrogen exchange data on short model peptides (r = 0.85) and the calculated hydration of the peptide backbone (r = 0.88). These results combined with the changes in enthalpy and entropy of unfolding of ubiquitin variants suggest that dehydration of the peptide backbone plays a significant role in defining the thermodynamic propensity scale at the C' position of the C-capping box in alpha-helices. This propensity scale is useful for protein secondary structure predictions and protein design.

Amino Acid Substitution↗
PubMed1991

Relative differences in the binding free energies of human immunodeficiency virus 1 protease inhibitors: a thermodynamic cycle-perturbation approach.

Peptidomimetic inhibitors of the human immunodeficiency virus 1 protease show considerable promise for treatment of AIDS. We have, therefore, been seeking computer-assisted drug design methods to aid in the systematic design of such inhibitors from a lead compound. Here we report thermodynamic cycle-perturbation calculations (using molecular dynamics simulations) to compute the relative difference in free energy of binding that results when one entire residue (valine) is deleted from one such inhibitor. In particular, we studied the "alchemic" mutation of the inhibitor Ac-Ser-Leu-Asn-(Phe-Hea-Pro)-Ile-Val-OMe (S1) to Ac-Ser-Leu-Asn-(Phe-Hea-Pro)-Ile-OMe (S2), where Hea is hydroxyethylamine, in two different (R and S) diastereomeric configurations of the hydroxyethylene group. The calculated (averaged for R and S) difference in binding free energy [3.3 +/- 1.1 kcal/mol (mean +/- SD); 1 cal = 4.184 J] is in good agreement with the experimental value of 3.8 +/- 1.3 kcal/mol, obtained from the measured Ki values for an equilibrium mixture of R and S configurations. Precise testing of our predictions will be possible when binding data become available for the two disastereomers separately. The observed binding preference for S1 is explained by the stronger ligand-protein interaction, which dominates an opposing contribution arising from the large desolvation penalty of S1 relative to S2. This calculation suggests that the thermodynamic cycle-perturbation approach can be useful even when a relatively large change in the ligand is simulated and supports the use of the thermodynamic cycle-perturbation algorithm for screening proposed derivatives of a lead inhibitor/drug prior to their synthesis.

Amino Acid Sequence↗
PubMed1991

The contribution of DNA single-stranded order to the thermodynamics of duplex formation.

We report a direct determination of the thermodynamic contribution that DNA single-stranded order makes to DNA duplex formation. By using differential scanning calorimetry (DSC) and temperature-dependent UV absorbance spectroscopy, we have characterized thermodynamically the thermally induced disruption of the 13-mer duplex [d(CGCATGAGTACGC)].[d(GCGTACTCATGCG)] (henceforth called S1.S2) and its component single strands, [d(CGCATGAGTACGC)] (henceforth called S1) and [d(GCGTACTCATGCG)] (henceforth called S2). These spectroscopic and calorimetric measurements yield the following thermodynamic profiles at 25 degrees C: delta G degree = 20.0 kcal/mol, delta H degree = 117.0 kcal/mol, and delta S degree = 325.4 cal.degree-1.mol-1 for duplex melting of S1.S2; delta G degree = 0.45 kcal/mol, delta H degrees = 29.1 kcal/mol, and delta S degree = 96.1 cal.degree-1.mol-1 for single-strand melting of S1; delta G degree = 1.44 kcal/mol, delta H degree = 27.2 kcal/mol, and delta S degree = 86.4 cal.degree-1.mol-1 for single-strand melting of S2 (1 cal = 4.184 J). These data reveal that the two single-stranded structures S1 and S2 are only marginally stable at 25 degrees C, despite exhibiting rather substantial transition enthalpies. This behavior results from enthalpy and entropy contributions of similar magnitudes that compensate each other, thereby giving rise to relatively small free energies of stabilization for the single strands at 25 degrees C. By contrast, the S1.S2 duplex state is very stable at 25 degrees C since the favorable transition entropy associated with duplex disruption (325.4 cal.degree-1.mol-1) is more than compensated for by the extremely large duplex transition enthalpy (117.0 kcal/mol). We also measured directly an enthalpy change (delta H degree) of -56.4 kcal/mol for duplex formation at 25 degrees C using isothermal batch-mixing calorimetry. This duplex formation enthalpy of -56.4 kcal/mol at 25 degrees C is very different in magnitude from the duplex disruption enthalpy of 117.0 kcal/mol measured at 74 degrees C by DSC. Since the DSC measurement reveals the net transition heat capacity change to be close to zero, we interpret this large disparity between the enthalpies of duplex disruption and duplex formation as reflecting differences in the single-stranded structures at 25 degrees C (the initial states in the isothermal mixing experiment) and the single-stranded structures at approximately 80 degrees C (the final states in the DSC experiment). In fact, the enthalpy for duplex formation at 25 degree C (-56.4 kcal/mol) can be combined with the sum of the integral enthalpies requires to melt each single strand from 25 to 80 degree C (23.6 kcal/mol for S1 and 27.2 kcal/mol for S2) to calculate a delta H degree of -107.2 kcal/mol for the hypothetical process of duplex formation from "random-coil" "unstacked" single strands at 25 degree C. The magnitude of this predicted delta H degree value for duplex formation is in good agreement with the corresponding parameter we measure directly by DSC for duplex disruption (117.0 kcal/mol), thereby lending credence to our interpretation and analysis of the data. Thus, our results demonstrate that despite being only marginally stable at 25 degree C, single strands can exhibit intramolecular interactions that enthalpically poise them for duplex formation. For the duplex studied herein, prior to association at 25 degree C, the two complementary single strands already possess > 40% of the total enthalpy (50.8/117) that ultimately stabilizes the final duplex state. This feature of single-stranded structure near room temperature can reduce significantly the enthalpic driving force one might predict for duplex formation from nearest-neighbor data, since such data generally are derived from measurements in which the single strands are in their random-coil states. Consequently, potential contributions from single-stranded structure must be recognized and accounted for when designing hybridization experiments and when using isothermal titration and/or batch mixing techniques to study the formation of duplexes and higher-order DNA structures (e.g., triplexes, tetraplexes, etc.) from their component single strands.

Calorimetry, Differential Scanning↗
PubMed1992

Thermodynamics of Cro protein-DNA interactions.

Using a highly sensitive pulsed-flow microcalorimeter, we have measured the changes in enthalpy and determined the thermodynamic parameters delta H, delta S degree, delta G degree, and delta C(p) for Cro protein-DNA association reactions. The reactions studied include sequence-nonspecific DNA association and sequence-specific DNA associations involving single- and multiple-base alterations and/or single-amino acid alteration mutants. (i) The association of Cro protein with nonspecific DNA at 15 degrees C is characterized by delta H = +4.4 kcal.mol-1 (1 cal = 4.18J), delta S degrees = 49 cal.mol-1.K-1, delta G degrees = -9.7 kcal.mol-1, and delta Cp congruent to 0; the association with specific high-affinity operator OR3 DNA is characterized by delta H = +0.8 kcal.mol-1, delta S degree = 59 cal.mol-1.K-1, delta G degree = -16.1 kcal.mol-1, and delta Cp = -360 cal.mol-1.K-1, respectively. Both nonspecific and specific Cro-DNA associations are entropy-driven. (ii) Plots of delta H vs. delta Cp and delta S degree vs. delta Cp for the 20 association reactions studied fall into two correlation groups with linear slopes of +9.4 K and -20.5 K and of -0.03 and -0.14, respectively. These regression lines have common intercepts, at the delta H and delta S degree values of nonspecific association (where delta Cp congruent to 0). The results suggest that there are, at least, two distinct conformational subclasses in specific Cro-DNA complexes, stabilized by different combinations of enthalpic and entropic contributions. The delta G degree and delta Cp values form an approximately single linear correlation group as a consequence of compensatory contributions from delta H and delta S degree to delta G degree and to delta Cp. Cro protein-DNA associations share some similar thermodynamic properties with protein folding, but their overall energetics are quite different. Although the nonspecific complex is stabilized predominantly by electrostatic forces, it appears that H bonds, van der Waals contacts, hydrophobic effects, and charge interactions all contribute to the stability (delta G degree and delta Cp) of the specific complex. (iii) The variations in the values of the thermodynamic parameters are in general accord with our knowledge of the structure of the Cro-DNA complex.

Base Sequence↗
PubMed1996

Thermodynamics of colchicinoid-tubulin interactions. Rrol of B-ring and C-7 substituent.

The quenching of tryptophan fluorescence has been used to determine the kinetic and thermodynamic parameters of binding of B-ring analogs of colchicine to tubulin. The on rate, activation energy, off-rate, and thermodynamics of binding reaction have been found to be controlled at different points of analog structure. The on-rate and off-rate of deacetamidocolchicine (DAAC) binding with tubulin is 17 times slower than that of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone-tubulin (AC-tubulin) interaction, although both reactions have very similar activation energies. The presence of B-ring alone does not significantly affect the thermodynamics of the binding reactions either, since both AC-tubulin and DAAC-tubulin interactions are enthalpy driven. Introduction of a NH2 group at C-7 position of the B-ring, as in deacetylcolchicine (NH2-DAAC) lowers the on-rate further with a significant rise in the value of the activation energy. However, bulkier substitutions at the same position, as in demecolcine (NHMe-DAAC) and N-methyldemecolcine (NMe2-DAAC) have no significant additional effect either on the on-rate or on the value of activation energy. Introduction of NH2 group in the C-7 position of B-ring also increases the positive entropy of the binding reaction to a significant extent, and it is maximum when NMe2 is substituted instead of NH2 group. Thus, interaction of NH2-DAAC, NHMe-DAAC, and NMe2-DAAC with tubulin are entropy driven. Our results suggest that the B-ring side chain of aminocolchicinoids makes contact(s) with dimeric tubulin molecules.

Animals↗
PubMed1997

Site-directed mutagenesis of either the highly conserved Trp-22 or the moderately conserved Trp-95 to a large, hydrophobic residue reduces the thermodynamic stability of a spectrin repeating unit.

As reported previously (MacDonald, R. I., Musacchio, A., Holmgren, R. A., and Saraste, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1299-1303), an unfolded peptide was obtained by site-directed mutagenesis of Trp-22 to Ala in the cloned, wild type 17th repeating unit (alpha17) of chicken brain alpha-spectrin. Trp occurs in position 22 of nearly all repeating units of spectrin. In the present study, Trp-22 was mutated to Phe or to Tyr to compare thermodynamic stabilities of urea-induced unfolding of alpha16 and mutants thereof. alpha16 was chosen for this study instead of alpha17, because alpha16 has two tryptophans, allowing urea-induced unfolding to be tracked by the fluorescence of the Trp remaining in each mutant peptide and by circular dichroism in the far UV. The free energies of unfolding of W22Y and W22F were 50% that of alpha16, showing that Trp-22 is crucial in stabilizing the triple helical bundle motif of the spectrin repeating unit. Mutation of the moderately conserved Trp-95 of alpha16 to Val, which occupies position 95 in alpha17, also yielded a peptide with 50% of the free energy of unfolding of alpha16. Thus, the thermodynamic stability of a given spectrin repeating unit may depend on both moderately and highly conserved tryptophans. Different structural roles of Trp-22 and Trp-95 in alpha16 are suggested by the slightly higher wavelength of maximum emission of Trp-22, the greater acrylamide quenching of Trp-95 than Trp-22, and the longer lifetime of Trp-95. For comparison with alpha16, urea-induced unfolding of spectrin dimer isolated from human red cells was monitored by far UV-CD and by tryptophan fluorescence. Thermodynamic parameters could not be rigorously derived for the stability of spectrin dimer because unfolding of spectrin dimer involved more than two states, unlike unfolding of cloned repeating units. However, the similar midpoints of CD-monitored denaturation curves of alpha16 and spectrin dimer, i. e. 2.7 and 3.2 M urea, respectively, indicate that investigation of cloned repeating units of spectrin can provide physiologically relevant information on these structures.

Amino Acid Sequence↗
PubMed1998

Thermodynamics of fatty acid binding to engineered mutants of the adipocyte and intestinal fatty acid-binding proteins.

We constructed 18 single amino acid mutants of the adipocyte fatty acid-binding protein (A-FABP) and 17 of the intestinal fatty acid-binding protein (I-FABP), at locations in the fatty acid (FA) binding sites. For each mutant protein, we measured thermodynamic parameters that characterize FA binding. Binding affinities ranged from about 200-fold smaller to 30-fold larger than the wild type (WT) proteins. Thermodynamic parameters revealed that binding affinities often inaccurately reported changes in protein-FA interactions because changes in the binding entropy and enthalpy were usually compensatory and larger than the binding free energy. FA-FABP interactions were quite different for I-FABP and A-FABP proteins. Binding affinities were larger and decreased to a greater degree with increasing FA solubility for most of the I-FABP as compared with the A-FABP proteins, consistent with a more hydrophobic binding site for the I-FABP proteins. In A-FABP, Ala substitutions for Arg106 and Arg126, which interact with the FA carboxylate, reduce affinities by about 100-fold, but in I-FABP, R106A increases affinities up to 30-fold. Moreover, in A-FABP, the thermodynamic parameters predict that the FA carboxylate location switches from the 126-position in R106A to the 106 position in R126A. Finally, the A-FABP proteins, in contrast to the I-FABP proteins, reveal significant heat capacity changes (DeltaCp) upon FA binding, and substitutions at residues Arg106 and Arg126 reduce the magnitude of DeltaCp.

Adipocytes↗
PubMed2000

Thermodynamic modulation of light chain amyloid fibril formation.

To obtain further insight into the pathogenesis of amyloidosis and develop therapeutic strategies to inhibit fibril formation we investigated: 1) the relationship between intrinsic physical properties (thermodynamic stability and hydrogen-deuterium (H-D) exchange rates) and the propensity of human immunoglobulin light chains to form amyloid fibrils in vitro; and 2) the effects of extrinsically modulating these properties on fibril formation. An amyloid-associated protein readily formed amyloid fibrils in vitro and had a lower free energy of unfolding than a homologous nonpathological protein, which did not form fibrils in vitro. H-D exchange was much faster for the pathological protein, suggesting it had a greater fraction of partially folded molecules. The thermodynamic stabilizer sucrose completely inhibited fibril formation by the pathological protein and shifted the values for its physical parameters to those measured for the nonpathological protein in buffer alone. Conversely, urea sufficiently destabilized the nonpathological protein such that its measured physical properties were equivalent to those of the pathological protein in buffer, and it formed fibrils. Thus, fibril formation by light chains is predominantly controlled by thermodynamic stability; and a rational strategy to inhibit amyloidosis is to design high affinity ligands that specifically increase the stability of the native protein.

Amino Acid Sequence↗
PubMed2000

Thermodynamic binding studies of lectins from the diocleinae subtribe to deoxy analogs of the core trimannoside of asparagine-linked oligosaccharides.

Lectins from seven different species of the Diocleinae subtribe have been recently isolated and characterized in terms of their carbohydrate binding specificities (Dam, T. K., Cavada, B. S., Grangeiro, T. B., Santos, C. F., de Sousa, F. A. M., Oscarson, S., and Brewer, C. F. (1998) J. Biol. Chem. 273, 12082-12088). The lectins included those from Canavalia brasiliensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. All of the lectins exhibited specificity for Man and Glc residues, but much higher affinities for the branched chain trimannoside, 3,6-di-O-(alpha-d-mannopyranosyl)-d-mannose, which is found in the core region of all asparagine-linked carbohydrates. In the present study, isothermal titration microcalorimetry is used to determine the binding thermodynamics of the above lectins, including a new lectin from Canavalia grandiflora, to a complete series of monodeoxy analogs of the core trimannoside. From losses in the affinity constants and enthalpies of binding of certain deoxy analogs, assignments are made of the hydroxyl epitopes on the trimannoside that are involved in binding to the lectins. The pattern of binding of the deoxy analogs is similar for all seven lectins, and similar to that of concanavalin A which is also a member of the Diocleinae subtribe. However, differences in the magnitude of the thermodynamic binding parameters of the lectins are observed, even though the lectins possess conserved contact residues in many cases, and highly conserved primary sequences. The results indicate that non-contact residues in the lectins, even those distant from the binding sites, modulate their thermodynamic binding parameters.

Amino Acid Sequence↗
PubMed2000

2'-O,4'-C-methylene bridged nucleic acid modification promotes pyrimidine motif triplex DNA formation at physiological pH: thermodynamic and kinetic studies.

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in an artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is, therefore, crucial in improving its therapeutic potential. To this end, we have investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA modification of TFO increased the binding constant of the pyrimidine motif triplex formation at neutral pH by approximately 20 times. The number and position of the 2',4'-BNA modification introduced into the TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity itself of the 2',4'-BNA-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant at neutral pH. Kinetic data demonstrated that the observed increase in the binding constant at neutral pH by the 2',4'-BNA modification of TFO resulted from the considerable decrease in the dissociation rate constant. Our results certainly support the idea that the 2',4'-BNA modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

DNA↗
PubMed2001

Thermodynamics of Ras/effector and Cdc42/effector interactions probed by isothermal titration calorimetry.

Proliferation, differentiation, and morphology of eucaryotic cells is regulated by a large network of signaling molecules. Among the major players are members of the Ras and Rho/Rac subfamilies of small GTPases that bind to different sets of effector proteins. Recognition of multiple effectors is important for communicating signals into different pathways, leading to the question of how an individual GTPase achieves tight binding to diverse targets. To understand the observed specificity, detailed information about binding energetics is expected to complement the information gained from the three-dimensional structures of GTPase/effector protein complexes. Here, the thermodynamics of the interaction of four closely related members of the Ras subfamily with four different effectors and, additionally, the more distantly related Cdc42/WASP couple were quantified by means of isothermal titration calorimetry. The heat capacity changes upon complex formation were rationalized in light of the GTPase/effector complex structures. Changes in enthalpy, entropy, and heat capacity of association with various Ras proteins are similar for the same effector. In contrast, although the structures of the Ras-binding domains are similar, the thermodynamics of the Ras/Raf and Ras/Ral guanine nucleotide dissociation stimulator interactions are quite different. The energy profile of the Cdc42/WASP interaction is similar to Ras/Ral guanine nucleotide dissociation stimulator, despite largely different structures and interface areas of the complexes. Water molecules in the interface cannot fully account for the observed discrepancy but may explain the large range of Ras/effector binding specificity. The differences in the thermodynamic parameters, particularly the entropy changes, could help in the design of effector-specific inhibitors that selectively block a single pathway.

Animals↗