SEARCH · Search PubMed

Results for “THERMODYNAMICS”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,027 records · Page 57Linked to original sources

PubMed2006

Redox thermodynamics of the ferric-ferrous couple of wild-type synechocystis KatG and KatG(Y249F).

Crystal structures and mass spectrometric analyses of catalase-peroxidases (KatGs) from different organisms revealed the existence of a peculiar distal Met-Tyr-Trp cross-link. The adduct appears to be important for the catalase but not the peroxidase activity of bifunctional KatG. To examine the effect of the adduct on enzyme redox properties and functions, we have determined the thermodynamics of ferric reduction for wild-type KatG and KatG(Y249F), whose tyrosine-to-phenylalanine mutation prevents cross-link formation. At 25 degrees C and pH 7.0, the reduction potential of wild-type KatG is found to be -226 +/- 10 mV, remarkably lower than the published literature values. The reduction potential of KatG(Y249F) is very similar (-222 +/- 10 mV), but variable temperature experiments revealed compensatory differences in reduction enthalpies and entropies. In both proteins, the oxidized state is enthalpically stabilized over the reduced state, but entropy is lost on reduction, which is in strong contrast to horseradish peroxidase, which also features a much more pronounced enthalpic stabilization of the ferriheme. With both proteins, the midpoint potential increased linearly with decreasing pH. We discuss whether the observed redox thermodynamics reflects the differences in structure and function between bifunctional KatG and monofunctional peroxidases.

Bacterial Proteins↗

PubMed2006

Thermodynamics of protein-protein interactions of cMyc, Max, and Mad: effect of polyions on protein dimerization.

The Myc-Max-Mad network of proteins activates or represses gene transcription depending on whether the dimerization partner of Max is c-Myc or Mad. To elucidate the physical properties of these protein-protein interactions, fluorescence anisotropy of TRITC-labeled Max was used. The binding affinities and thermodynamics of dimerization of the Max-Max homodimer and c-Myc-Max and Mad-Max heterodimers were determined. Our results indicate that c-Myc and Max form the most stable heterodimer. Previous work [Kohler, J. J., Metallo, S. J., Schneider, T. L., and Schepartz, A. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 11735-9] has shown that instead of dimerizing first and then binding to DNA, these proteins use a monomer pathway in which a monomer binds to DNA followed by dimerization on the surface of the DNA. The DNA E-box affects the dimerization, but nonspecific effects may also play a role. The influence of polyions, poly-L-lysine and poly-L-glutamic acid, were investigated to determine the effects of charged polymers other than DNA on homodimerization and heterodimerization. While the positively charged poly-L-lysine, PLL, did not show any significant effect, negatively charged poly-L-glutamic acid, PLG, stabilized both heterodimers and homodimers by 2-3 kJ/mol. These data suggest that in the cell nucleus the presence of negatively charged DNA or RNA could nonspecifically aid in association of these proteins. Calculations of DeltaH degrees and DeltaS degrees from the temperature dependence of K(d) indicated that although the thermodynamic parameters for the dimer are different, the reactions for all three dimers are driven by negative (favorable) enthalpic and negative (unfavorable) entropic contributions. In the presence of PLG, entropy became more negative with the effect being largest for c-Myc-Max heterodimers. This suggests that van der Waals and H-bonding interactions are predominant in dimerization of these proteins.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed2006

A unique molten globule state occurs during unfolding of cytochrome c by LiClO4 near physiological pH and temperature: structural and thermodynamic characterization.

We have carried out denaturation studies of bovine cytochrome c (cyt c) by LiClO4 at pH 6.0 and 25 degrees C by observing changes in difference molar absorbance at 400 nm (Deltaepsilon400), mean residue ellipticities at 222 nm ([theta]222) and difference mean residue ellipticity at 409 nm (Delta[theta]409). The denaturation is a three-step process when measured by Deltaepsilon400 and Delta[theta]409, and it is a two-step process when monitored by [theta]222. The stable folding intermediate state has been characterized by near- and far-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements. A comparison of the conformational and thermodynamic properties of the LiClO4-induced molten globule (MG) state with those induced by other solvent conditions (e.g., low pH, LiCl, and CaCl2) suggests that LiClO4 induces a unique MG state, i.e., (i) the core in the LiClO4-induced state retains less secondary and tertiary structure than that in the MG states obtained in other solvent conditions, and (ii) the thermodynamic stability associated with the LiClO4-induced process, native state <--> MG state, is the same as that observed for each transition between native and MG states induced by other solvent conditions.

Animals↗

PubMed2006

A thermodynamic comparison of HPr proteins from extremophilic organisms.

A thermodynamic stability study of five histidine-containing phosphocarrier protein (HPr) homologues derived from organisms inhabiting diverse environments is described. These HPr homologues are from Bacillus subtilis (Bs), Streptococcus thermophilus (St), Bacillus staerothermophilus (Bst), Bacillus halodurans (Bh), and Oceanobacillus iheyensis (Oi). Analyses of solvent and thermal denaturation experiments provide the cardinal thermodynamic parameters, like deltaG, deltaH, deltaS, T(m), and deltaC(p), that characterize the conformational stability for each homologue. The homologue from Bacillus staerothermophilus (BstHPr) was established as the most thermostable homologue and also the homologue with highest deltaG at all temperatures. A good correlation between habitat temperature of the organism and thermal stability of the protein is also seen. Stability curves (deltaG vs T) for every homologue are also reported; these reveal very similar deltaC(p) and temperature of maximum stability (T(S)) values for all HPr homologues. Stability curves show that the higher thermal stability of some homologues is not a result of change in curvature of the curve or a shift to higher temperature, but rather a displacement of the stability curves to higher deltaG values. Stability curves also allowed estimation of deltaG at habitat temperature of the organisms, and we find good agreement between homologues. Electrostatic contributions to stability of each homologue were investigated by measuring stability as a function of varying pH and NaCl concentration, and our results suggest that most HPr homologues share similar electrostatic contributions to stability.

Amino Acid Sequence↗

PubMed2006

Thermodynamics of phosphopeptide binding to the human peptidyl prolyl cis/trans isomerase Pin1.

Proteins containing phosphorylated Ser/Thr-Pro motifs play key roles in numerous regulatory processes in the cell. The peptidyl prolyl cis/trans isomerase Pin1 specifically catalyzes the conformational transition of phosphorylated Ser/Thr-Pro motifs. Here we report the direct analysis of the thermodynamic properties of the interaction of the PPIase Pin1 with its substrate-analogue inhibitor Ac-Phe-D-Thr(PO3H2)-Pip-Nal-Gln-NH2 specifically targeted to the PPIase active site based on the combination of isothermal titration calorimetry and studies on inhibition of enzymatic activity of wt Pin1 and active site variants. Determination of the thermodynamic parameters revealed an enthalpically and entropically favored interaction characterized by binding enthalpy deltaH(ITC) of -6.3 +/- 0.1 kcal mol(-1) and a TdeltaS(ITC) of 4.1 +/- 0.1 kcal mol(-1). The resulting dissociation constant KD for binding of the peptidic inhibitor with 1.8 x 10(-8) M resembles the dissociation constant of a Pin1 substrate in the transition state, suggesting a transition state analogue conformation of the bound inhibitor. The strongly decreased affinity of Pin1 for ligand at increasing ionic strength implicates that the potential of bidentate binding of a substrate protein by the PPIase and the WW domain of Pin1 may be required to deploy improved efficiency and specificity of Pin1 under conditions of physiological ionic strength.

Binding Sites↗

PubMed2006

Thermodynamic analysis of the binding of oxidized and reduced FMN cofactor to Vibrio harveyi NADPH-FMN oxidoreductase FRP apoenzyme.

The Vibrio harveyi NADPH-specific flavin reductase FRP follows a ping-pong mechanism but switches to a sequential mechanism in the luciferase-coupled reaction. The bound FMN co-isolated with FRP, while acting as a genuine cofactor in the single-enzyme reaction, functions in the luciferase-coupled reaction as a prebound substrate and is directly transferred to luciferase once it is reduced [Lei, B., and Tu, S.-C. (1998) Biochemistry 37, 14623-14629]. With the aim of better understanding the functions of FMN in the FRP holoenzyme, this study was undertaken to quantify and compare the thermodynamic properties of the binding of oxidized and reduced FMN by the FRP apoenzyme. By isothermal titration calorimetry (ITC) measurements in various buffers at pH 7.0 and 15-30 degrees C, the binding of FMN by apo-FRP was found to be noncooperative, exothermic, and primarily enthalpy driven. The binding free energy change (hence, the association constant) was nearly invariant over this temperature range. Significant conformational changes in FRP upon binding of FMN were indicated. Equilibrium bindings of reduced flavins by flavin-dependent proteins have rarely been studied. In this work, the thermodynamic properties of binding of reduced FMN by apo-FRP were found to closely resemble those of FMN binding under three sets of experimental conditions via ITC measurements and, in one case, fluorescence quenching. The kinetically deduced ping-pong mechanism of FRP is now supported by direct measurements of binding affinities of the oxidized and reduced FMN cofactors. These findings are also discussed in relation to the function of FRP as a reduced flavin donor in the FRP-luciferase couple.

Apoenzymes↗

PubMed2006

Thermodynamic basis for redox regulation of the Yap1 signal transduction pathway.

The Yap1 oxidative stress signal transduction pathway found in Saccharomyces cerevisiae is redox-regulated. We have examined the thermodynamic basis of the disulfide/dithiol couples that are involved in the regulation of this pathway. The oxidized form of the Yap1 redox domain (Yap1-RD) fragment, derived from the Yap1 transcription factor, contains two disulfide bonds, one between Cys303 and Cys598 and one between Cys310 and Cys629. Oxidation-reduction titrations reveal the presence of two separate two-electron redox couples in Yap1-RD, with redox midpoint potentials (E(m)) of -155 and -330 mV, respectively, at pH 7.0. We measured E(m) values of -275 and -265 mV for the two cytoplasmic S. cerevisiae thioredoxins, Trx1 and Trx2, respectively, both at pH 7.0. Last, we measured an E(m) value of -255 mV for the Cys36-Cys82 disulfide bond at pH 6.0 in the glutathione peroxidase-like enzyme, oxidant receptor protein (Orp1). We were unable to obtain satisfactory redox titration data for Orp1 at pH 7.0, but if the redox-active disulfide of Orp1 exhibits the -59 mV per pH unit dependence for E(m) typical of protein disulfides in this pH region, an E(m) value of -315 mV can be estimated for Orp1 at pH 7.0 by extrapolation. Together, these data suggest that, at physiological ratios of Trx(ox)/Trx(red), the reduction of both the E(m) = -315 mV disulfide of Orp1 and the E(m) = -330 mV disulfide of Yap1 by either Trx1 or Trx2 would be thermodynamically possible.

Amino Acid Sequence↗

PubMed2006

Thermodynamics of the ATPase cycle of GlcV, the nucleotide-binding domain of the glucose ABC transporter of sulfolobus solfataricus.

ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains (NBDs) that energize the transport reaction. Binding of ATP to the NBD monomer results in formation of a NBD dimer. Hydrolysis of the ATP drives the dissociation of the dimer. The thermodynamics of distinct steps in the ATPase cycle of GlcV, the NBD of the glucose ABC transporter of the extreme thermoacidophile Sulfolobus solfataricus, were studied by isothermal titration calorimetry using the wild-type protein and two mutants, which are arrested at different steps in the ATP hydrolytic cycle. The G144A mutant is unable to dimerize, while the E166A mutant is defective in dimer dissociation. The ATP, ADP, and AMP-PNP binding affinities, stoichiometries, and enthalpies of binding were determined at different temperatures. From these data, the thermodynamic parameters of nucleotide binding, NBD dimerization, and ATP hydrolysis were calculated. The data demonstrate that the ATP hydrolysis cycle of isolated NBDs consists of consecutive steps where only the final step of ADP release is energetically unfavorable.

ATP-Binding Cassette Transporters↗

PubMed2007

Thermodynamic characterization of the interaction between CAR-RXR and SRC-1 peptide by isothermal titration calorimetry.

The constitutive androstane receptor (CAR) enhances transcription of specific target genes that regulate several metabolic pathways. CAR functions as an obligate heterodimer (CAR-RXR) with the retinoid X receptor (RXR). Also part of the active receptor complex is the steroid receptor coactivator-1 (SRC-1) which interacts with the receptor complex via specific receptor interaction domains (RIDs). A peptide derived from SRC-1 RID2 is used to study the thermodynamic properties of the interaction with the CAR-RXR ligand binding domain (LBD) complex. In the absence of ligands for both CAR and RXR, binding of coactivator peptide to the CAR-RXR heterodimer is characterized by a favorable enthalpy change and an unfavorable entropy change. The addition of the CAR agonist, TCPOBOP, increases the affinity for coactivator by decreasing the unfavorable entropy and increasing the favorable intrinsic enthalpy of the interaction. The RXR ligand, 9-cis-RA, generates a second SRC-1 site and increases the affinity by improving the entropic component of binding. There is an additional increase in affinity for one of the two sites in the presence of both ligands. The change in heat capacity (deltaCp) is also investigated. A 2-fold difference in deltaCp is observed between liganded and unliganded CAR-RXR. The observed thermodynamic parameters for binding of SRC-1 peptide to liganded and apo CAR-RXR as well as the difference in the deltaCp data provide evidence that the apo CAR-RXR heterodimer is conformationally mobile. The more favorable enthalpic contribution for TCPOBOP-bound CAR-RXR indicates that preformation of the binding site improves the complementarity of the coactivator-receptor interaction.

Alitretinoin↗

PubMed1996

Interactions of aristololactam beta-D-glucoside with right-handed and left-handed forms of synthetic deoxyribonucleic acid: spectroscopic and thermodynamic study.

The interaction of aristololactam beta-D-glucoside (ADG) with different polymorphic structures of poly(dG-me5dC).poly(dG-me5dC), poly(dG-dC).poly(dG-dC), and poly(dI-dC). poly(dI-dC) has been studied by spectrophotometric, spectrofluorimetric, circular dichroism, UV melting profiles, and thermodynamic analysis. The binding of ADG to B-form duplexes is characterized by the typical bathochromic and hypochromic effects in the absorption spectrum, quenching of steady-state fluorescence intensity, a decrease in fluorescence quantum yield of ADG, an increase in fluorescence polarization anisotropy, an increase of thermal transition temperature, and perturbation in circular dichroic spectrum. Scatchard analysis indicates that ADG binds to the right-handed form of each polymer in a noncooperative manner. Comparative binding parameters determined from absorbance titration by Scatchard analysis, employing the excluded site model, indicate a stronger binding of ADG to the B-form of poly(dG-me5-dC). poly(dG-me5dC) than to the B-form of poly(dG-dC).poly(dG-dC) or poly(dI-dC). poly(dI-dC). Thermodynamic parameters (delta G degree, delta H degree, and delta S degree) obtained by van't Hoff analysis of the data show that the process of binding to all B-form duplexes is exothermic and enthalpy driven as characterized by a favorable negative enthalpy change (delta H degree). The binding is opposed by a negative entropy change (delta S degree) contribution. Conformational changes indicate that the alkaloid converts the left-handed form of poly(dG-dC). poly(dG-dC), and its methylated analogue and high salt form of poly(dI-dC). poly(dI-dC) to a bound right-handed form, while it inhibits both the rate and extent of the B to Z transition. These studies reveal that ADG binds strongly to B-form polymers while it does not bind to polymers of Z-form.

Alkaloids↗

PubMed1996

Thermodynamic studies of the core histones: pH and ionic strength effects on the stability of the (H3-H4)/(H3-H4)2 system.

The self-associative behavior and the thermal stability of the H3/H4 histone complex was studied in low-ionic strength conditions by several physicochemical techniques, including differential scanning calorimetry and circular dichroism spectroscopy. At neutrality, the major molecular species present in solution is the (H3-H4)2 tetramer. Its thermodynamic properties cannot be studied directly though, since its thermal denaturation is completely irreversible even at the lowest salt concentrations. However, a complete thermodynamic analysis can be performed at low ionic strength and pH 4.5, where the (H3-H4)2 tetramer is quantitatively dissociated into two H3-H4 dimers and where almost complete reversibility of the thermal transitions is attained. The unfolding transition temperature of the 26.5 kDa H3-H4 dimer increases as a function of both the ionic strength of the solvent and the total protein concentration. The thermal denaturation of the H3-H4 dimer is characterized by the presence of a single calorimetric peak, centered at 58 degrees C, with a corresponding enthalpy change of 25 kcal/mol of a 13 kDa monomer unit and a change in heat capacity upon unfolding of about 0.6 kcal/(K mol of 13 kDa monomer unit). The complex between histones H3 and H4 (tetramer or dimer) is stable between pH 9.5 and 3.0. At pH 1.5, the system is almost completely unfolded at all temperatures. At low ionic strengths and pH values between 5.0 and 2.5, the H3-H4 dimer behaves as a highly cooperative system, melting as a single unit; i.e. individual H3 and H4 folded monomers are not detectable during the treatment. The two-state mechanism accounting for the unfolding of the H3-H4 dimer at pH 4.5 is the same as that described for the H2A-H2B dimer at neutrality. Just like for the H2A and H2B histones, the H3 and H4 polypeptides are properly folded only when assembled as H3-H4 dimers or in higher-order histone assemblies. Therefore, coupling along the interfaces of the two chains within the heterodimer is the major factor contributing to the stabilization of the secondary and tertiary structures of the chains as well as of the histone dimers.

Animals↗

PubMed1996

Thermodynamics of the unfolding and spectroscopic properties of the V66W mutant of Staphylococcal nuclease and its 1-136 fragment.

Spectroscopic studies have been performed to characterize the solution structure of the V66W mutant of Staphylococcal nuclease and the corresponding 1-136 fragment, referred to as V66W'. Whereas wild-type nuclease has a single tryptophan residue at position 140, the V66W mutant has a second tryptophan residue at position 66, which is the only such residue in V66W'. Steady-state and time-resolved fluorescence studies show Trp-66 in V66W' to have a blue emission, a relatively large fluorescence quantum yield, a long lifetime, a significant degree of protection from solute quenchers, and to depolarize with a relatively long rotational correlation time. These results characterize Trp-66 in V66W' as being a buried residue, which indicates that this fragment retains some global structure. Circular dichroism (CD) data are consistent with the fragment having lost most of the alpha-helical content of the wild type, while retaining beta-sheet structure. The CD spectrum in the aromatic region also suggests that Trp-66 in the fragment experiences an asymmetric environment, which is not identical to that in the full length mutant, V66W. In addition, optical detection of triplet state magnetic resonance (ODMR) spectroscopy can clearly resolve the tryptophan residues and demonstrates differences between the local environment of Trp-66 in V66W and in V66W', as well as small differences in the Trp-140 environment in wild type and in V66W. Guanidine-HCl induced and thermally induced unfolding studies were performed by simultaneously acquiring CD and fluorescence data as a function of the perturbation and then performing a global analysis of such multiple data sets in terms of two-state and three-state unfolding models. Whereas data for wild-type nuclease and the V66W' fragment are well characterized by a two-state unfolding model, data for the V66W mutant are better characterized by a three-state process. That is, both the denaturant- and temperature-induced unfolding of V66W involves the significant population of an equilibrium unfolding intermediate. Our global analyses yield thermodynamic parameters for the unfolding transitions, and we show that the data for V66W can be described by a constrained three-state model in which the transition of the intermediate to the fully unfolded state is fixed to have the same thermodynamic parameters that describe the unfolding of the V66W' fragment.

Calorimetry↗

PubMed1996

Alanine point-mutations in the reactive region of bovine pancreatic trypsin inhibitor: effects on the kinetics and thermodynamics of binding to beta-trypsin and alpha-chymotrypsin.

In an effort to relate structural, kinetic, and thermodynamic features in a model macromolecular recognition process, the amino acid residues in the reactive surface of bovine pancreatic trypsin inhibitor (BPTI) and surrounding residues were substituted individually by alanine, and the effects of the point-mutations on the kinetics and thermodynamics of inhibition by BPTI toward trypsin and chymotrypsin were investigated. Fifteen alanine mutants were produced. The majority of the BPTI mutants exhibited a binding affinity similar to that of the wild-type protein. The exceptions were the primary specificity site (PI) mutant and those mutants that seem to have nonlocal perturbations of structure, as revealed by circular dichroism and thermostability measurements. The mutation at the P1 site caused a reduction in the binding free energy of 10 and 1.8 kcal mol-1 for trypsin and chymotrypsin, respectively. The losses in binding affinity were determined almost exclusively by an increase in the dissociation rate constant. However, the rate of association of the P1 mutant, Lys-15-Ala, with trypsin was also drastically reduced (> 200-fold). Calorimetric measurements of the heats of binding for the association of chymotrypsin with the wild-type inhibitor and its alanine mutants allowed determination of the relative contributions of the changes in enthalpy and entropy to the free energy of binding. Compensatory changes in the two parameters were observed in several cases, which were attributed to desolvation effects at the binding interface.

Alanine↗

PubMed1996

Thermodynamic characterization of the partially unfolded state of Ca(2+)-loaded bovine alpha-lactalbumin: evidence that partial unfolding can precede Ca2+ release.

The thermal denaturation of bovine alpha-lactalbumin (BLA) was studied at pH 7.5 and at various Ca2+ concentrations using near-UV circular dichroism and differential scanning calorimetry. The Ca2+ dependence of the denaturation equilibria proves that, in the transition region, partially unfolded alpha-lactalbumin consists of a mixture of Ca(2+)-loaded and Ca(2+)-free protein. The thermodynamic parameters of the unfolding of these two species were determined at 68 degrees C and were then compared with one other, with the thermodynamic parameters deduced from calorimetric titration of alpha-lactalbumin with Ca2+, and with those derived from Ca2+ titration of a mutant human lysozyme having an engineered Ca(2+)-binding site. This comparison indicated that (a) the unfolding curves for Ca(2+)-BLA deduced from the near-UV ellipticity change are more able to distinguish between unfolding with and without Ca2+ release than those deduced from differential scanning calorimetry, (b) the Ca(2+)-loaded denaturated state of BLA is more folded than the Ca(2+)-free protein at 68 degrees C, and (c) a heat-induced unfolding process, consisting of an initial Ca2+ release, followed by a conformational relaxation, is unlikely to occur at the experimental pH and in the millimolar region of Ca2+ concentrations, due to the large free energy requirement of the initial step. A more probable mechanism would be unfolding via a Ca(2+)-loaded intermediately unfolded state, with subsequent Ca2+ release.

Animals↗

PubMed1996

Nonpolar interactions of thrombin and its inhibitors at the fibrinogen recognition exosite: thermodynamic analysis.

Nonpolar interactions play a major role in the association of the fibrinogen recognition exosite of thrombin with the C-terminal fragment (55-65), Asp-Phe-Glu-IIe-Pro-Glu-Glu-Tyr-Leu-Gln, of hirudin, which is a naturally occurring thrombin inhibitor. The thermodynamic details (free energy, enthalpy, entropy, and heat capacity) of the molecular recognition are studied by using five analogs of a synthetic bivalent thrombin inhibitor (P552), tert-butylbenzensulfonyl-Arg-(D-pipecolic acid)-(12-amino-dodecanoic acid)-(gamma-aminobutyric acid)-hirudin55-65. The residue of PheH56, IleH59, ProH60, TyrH63, or LeuH64 in hirudin 55-65 segment is substituted by Gly in each analog in order to elucidate the contributions of these nonpolar side chains. The results show that the interactions of these nonpolar side chains with thrombin are enthalpy-driven, except for the contribution of the PheH56 side chain which is entropy-driven. Interestingly, molecular modeling predicts a large conformational change due to the Gly substitution of PheH56. In analyzing the correlation among the thermodynamic and structural properties of the nonpolar interaction, a good correlation is observed between the binding free energy and the hydrophobicity of the molecular surface; i.e., tighter binding is observed as more nonpolar atoms are buried and more polar atoms are exposed upon molecular association.

Antithrombins↗

PubMed1996

Thermodynamic and kinetic measurements of promoter binding by T7 RNA polymerase.

Previous steady state kinetic studies of the initiation of transcription by T7 RNA polymerase have shown that melting of the DNA helix near the transcription start site is not rate limiting [Maslak, M., & Martin, C. T. (1993) Biochemistry 32, 4281-4285]. In the current work, fluorescence changes in a nucleotide analog incorporated within the promoter are used to monitor changes in the DNA helix associated with polymerase binding. The fluorescence of 2-aminopurine has been previously shown to depend on the environment of the base, with fluorescence increasing in the transition from a double-stranded to a single-stranded environment [Xu, D., Evans, K.O., & Nordlund, T. M.(1994) Biochemistry 33, 9592-9599]. Fluorescence changes associated with polymerase binding to promoters incorporating 2-aminopurine at positions -4 through -1 support a model which includes melting, in the statically bound complex, of the region of the promoter near the start site. Equilibrium titrations at 25 degrees C with label at position -2 provide a thermodynamic measure of the dissociation constant (Kd = 4.8 nM) for promoter binding, while stopped-flow kinetic assays measure the apparent association (k1 = 5.6 x 10(7) M-1 s-1) and dissociation (k-1 = 0.20 s-1) rate constants for simple promoter binding (the ratio k-1/k1 = 3.6 nM, in good agreement with the thermodynamic measurement of Kd). These results suggest that binding is close to the diffusion-controlled limit and helix melting is extremely rapid. In studies of structurally altered promoters, a base functional group substitution at position -10 is shown to significantly decrease k1, with little effect on k-1. In contrast, removal of the nontemplate strand from position +1 downstream results in a large decrease in k-1, with no significant effect on k1.

DNA-Directed RNA Polymerases↗

PubMed1996

Interaction of pyridine nucleotide substrates with Escherichia coli dihydrodipicolinate reductase: thermodynamic and structural analysis of binary complexes.

E. coli dihydrodipicolinate reductase exhibits unusual nucleotide specificity, with NADH being kinetically twice as effective as NADPH as a reductant as evidenced by their relative V/K values. To investigate the nature of the interactions which determine this specificity, we performed isothermal titration calorimetry to determine the thermodynamic parameters of binding and determined the three-dimensional structures of the corresponding enzyme-nucleotide complexes. The thermodynamic binding parameters for NADPH and NADH were determined to be Kd = 2.12 microM, delta G degree = -7.81 kcal mol-1, delta H degree = -10.98 kcal mol-1, and delta S degree = -10.5 cal mol-1 deg-1 and Kd = 0.46 microM, delta G degree = -8.74 kcal mol-1, delta H degree = -8.93 kcal mol-1, and delta S degree = 0.65 cal mol-1 deg-1, respectively. The structures of DHPR complexed with these nucleotides have been determined at 2.2 A resolution. The 2'-phosphate of NADPH interacts electrostatically with Arg39, while in the NADH complex this interaction is replaced by hydrogen bonds between the 2' and 3' adenosyl ribose hydroxyls and Glu38. Similar studies were also performed with other pyridine nucleotide substrate analogs to determine the contributions of individual groups on the nucleotide to the binding affinity and enthalpic and entropic components of the free energy of binding, delta G degree. Analogs lacking the 2'-phosphate containing homologs. For all analogs, the total binding free energy can be shown to include compensating enthalpic and entropic contributions to the association constants. The entropy contribution appears to play a more important role in the binding of the nonphosphorylated analogs than in the binding of the phosphorylated analogs.

Amino Acid Sequence↗

PubMed1997

Thermodynamic properties of a conformationally constrained intramolecular DNA triple helix.

We describe the thermodynamic properties of an intramolecular triple helix with two all-thymine linker loops in which the Hoogsteen strand is covalently crosslinked to the underlying Watson-Crick hairpin duplex by means of a disulfide bridge. We compare these properties to those of the corresponding intramolecular triplex without the disulfide crosslink. Optical and calorimetric measurements reveal that the uncrosslinked parent triplex melts in a biphasic manner above pH 6, with the initial triplex to duplex transition (Hoogsteen strand release) occurring at lower temperatures than subsequent melting of the hairpin helix. By contrast, crosslinking increases the thermal stability of the Hoogsteen transition such that the triplex and underlying hairpin duplex melt as a single transition under all conditions studied. Model independent thermodynamic data obtained by differential scanning calorimetry reveals the crosslink-induced increase in triplex thermal stability corresponds to a free energy stabilization of about 3 kcal/mol, with this stabilization being entirely entropic in origin. In other words, the crosslink is enthalpically neutral, but nevertheless, induces a triplex stabilization of 3 kcal/mol due to a reduction in the entropy change associated with triplex melting. In an effort to define the origin(s) of this entropic impact, we measured the pH and ionic strength dependence of the melting transitions. From a comparison of the melting transitions at different pH values and ionic strengths, we estimate that 0.4 more protons are associated with the crosslinked triplex state than with the uncrosslinked triplex, and 1.3 fewer counterions are released on melting the crosslinked triplex. We discuss how such crosslink-induced changes in proton binding and counterion release, in conjunction with potential changes in hydration and conformational freedom, could combine to give rise to the observed changes in entropy.

Base Sequence↗