Isothermal Titration Microcalorimetry Research Articles

PEGylated Degradable Composite Nanoparticles Based on Mixtures of PEG-b-Poly(γ-benzyl l-glutamate) and Poly(γ-benzyl l-glutamate)

In the present work, the possibility to obtain PEGylated nanoparticles from two PBLG derivatives, PEG-b-poly(γ-benzyl L-glutamate), PBLG-PEG-60, and poly(γ-benzyl L-glutamate), PBLG-Bnz-50, by nanoprecipitation has been investigated. Particles were prepared not only from one polymer (PBLG-PEG-60 or PBLG-Bnz-50), but also from mixtures of two PBLG derivatives, PBLG-PEG-60 and PBLG-Bnz-50, in different ratios (90/10, 77/23, and 40/60 wt %). Because of the presence of PEG chains, hydrophilic particles were obtained, which was confirmed by ζ potential measurements (ζ from -13 mV and -21 mV) and by isothermal titration microcalorimetry (ITC). This last technique has shown no heat exchange when BSA was added to PEGylated nanoparticles. Further, complement activation has been evaluated by crossed immuno-electrophoresis demonstrating that the introduction of 77 wt % of PEGylated PBLG chains in the particles was enough to ensure a low complement activation activity. This effect was strongly correlated to the ζ potential of the particles, which decreased with an increase of PEG chains content. Interestingly, such properties are of interest for the preparation of degradable stealth nanocarriers. Moreover, it is suggested that the introduction of a reasonable amount (up to 20 wt %) of a second copolymer in the particle composition can be possible without modifying their stealth character. Moreover, the presence of this second copolymer would help to introduce a second functionality to the particles.

A concise analysis of the effect of temperature and propanediol-1, 2 on Pluronic F127 micellization using isothermal titration microcalorimetry

This article aims to explore the possibility of using isothermal titration microcalorimetry (ITC) for the investigation of F127 micellization and to study the effect of temperature and addition of propanediol-1,2 on F127 micellization behavior. From this work, ITC proved efficient to be used as a tool for the determination of the critical micellization concentration (CMC) and the enthalpy of micellization (Δ H mic) of F127, from which the other thermodynamic parameters were calculated (free energy (Δ G mic), entropy (Δ S mic), and heat capacity of micellization (Δ C p,mic)). The micellization of F127 was confirmed to be a strongly endothermic process with predominance of hydrophobic interactions ( TΔ S mic > Δ H mic) and the correlation of enthalpy and entropy of micelle formation exhibits an excellent linearity. The temperature dependence of F127 micellization was revealed by using ITC, since the CMCs values were, respectively, 0.197, 0.095, 0.085, and 0.079 mM for temperatures 28, 29, 30, and 31 °C. Secondly, by the addition of propanediol-1,2 to the micellization medium containing 1.187 mM of F127, the CMC was shifted to lower values (0.095, 0.081, 0.077, 0.069 and 0.066 mM, respectively, for propanediol-1,2 concentrations of 0%, 1.4%, 2.3%, 2.8%, and 3.7% w/v in the micellization medium). Finally, ITC was used as diagnostic tool with the aim of checking the reproducibility of the experiments independently on the kinetic and the dynamic aspects related to the micelle formation breakup. However, in this work we proved that the use of ITC for the determination of the CMC and thermodynamic parameters associated with F127 micellization is limited to a range of temperatures when sigmoidal curves were obtained.

Micelle Formation of Long-Chain Imidazolium Ionic Liquids in Aqueous Solution Measured by Isothermal Titration Microcalorimetry

Self-association of three long-chain imidazolium ionic liquids (ILs), [C12mim]Br, [C14mim]Br, and [C16mim]Br, in aqueous solution was studied by surface tension measurements over a temperature range from (278.15 to 328.15) K. Effects of temperature and hydrocarbon chain length of the three long-chain ILs on the critical micelle concentration (CMC) were examined. Thermodynamic parameters, ΔmicG, ΔmicH, and ΔmicS, of micellization were determined by applying a mass-action model equation. Isothermal titration microcalorimetry was used to obtain the enthalpy change upon micellization of the three long-chain ILs at 298.15 K. Moreover, the CMCs and the thermodynamic parameters (ΔmicG, ΔmicH, and ΔmicS) were determined based on the isothermal titration microcalorimetry results. These CMC values are approximately equal to the CMCs obtained by surface tension measurement.

Microcalorimetric investigation on the formation of supramolecular nanoassemblies of associative polymers loaded with gadolinium chelate derivatives

In this study, isothermal titration microcalorimetry (ITC) and molecular modeling were used to investigate the mechanism of formation of supramolecular nanoassemblies prepared by mixing aqueous solutions of two associative polymers (i.e. polymerised β-CD (pβ-CD) and dextran grafted with lauryl side chains (MD)). Their capacity to entrap a contrast agent for magnetic resonance imaging (a gadolinium (Gd 3+) derivative) has been determined by the same methods. ITC experiments have been employed to evaluate the stoichiometry of interaction ( N), association constants ( K) and thermodynamic parameter variation associated with complexation between hosts and guests involved in this system. The inclusion compounds studied were: as hosts, β-CD and pβ-CD, and as guests, MD, adamantyl amine, and a Gd 3+ complex functionalized with adamantane. It has been demonstrated that pβ-CD cavities tend to interact more favourably with MD ( K = 25,000 M −1) than with adamantly amine ( K = 3650 M −1) and Gd 3+ complex ( K = 1460 M −1), forming 1:1 complexes, as also confirmed by molecular modeling. Noteworthy, the Gd 3+ derivatives, although incorporated in the supramolecular nanoassemblies (by inclusion into the β-CD cavities of pβ-CD), did not destabilize the pβ-CD–MD inclusion complexes, probably because the interaction between pβ-CD and MD was stronger. Finally, the analysis of thermodynamic parameters revealed that the interaction between MD and pβ-CD was entropy driven (|Δ H| < | TΔ S|) while the interactions of adamantly amine and Gd 3+ complex with β-CD and pβ-CD were enthalpy driven and dominated by van der Walls forces (|Δ H| > | TΔ S|).

New Insights into Chitosan−DNA Interactions Using Isothermal Titration Microcalorimetry

The interaction of chitosan with plasmid DNA was investigated as a function of pH, buffer composition, degree of deacetylation (DDA), and molecular weight (M(n)) of chitosan, using isothermal titration microcalorimetry (ITC). The Single Set of Identical Sites model was used to obtain the enthalpy of interaction, the binding constant, and the stoichiometry of binding. The chitosan-DNA interaction was shown to be coupled with proton transfer from the buffer to chitosan, as revealed by the dependence of the measured heat release on the ionization enthalpy of the buffer. The measured enthalpy of binding was almost entirely due to proton transfer, because it was accounted for by the enthalpy of ionization of the buffer and of chitosan once the number of protons transferred was calculated. This proton transfer during binding resulted in the protonation of an additional 17, 37, and 58% of total glucosamine units at pH 5.5, 6.5, and 7.4, respectively. The strong polyanionic nature of DNA facilitates the ionization of glucosamines of chitosan upon complexation and is responsible for proton transfer. Interestingly, using the chitosan-DNA stoichiometry provided by ITC and the calculated degree of ionization of chitosan in the complex, the charge ratio of protonated amines to negative phosphate groups in the complex was nearly constant at 0.50-0.75 after saturation and was independent of the pH, buffer type and chitosan molecular characteristics. The chitosan-DNA binding constant was in the range of 10(9)-10(10) M(-1). The binding constant was pH-dependent and was greater at lower pH due to increased electrostatic attraction to DNA when chitosan is highly charged. Furthermore, the DDA and molecular weight of chitosan exerted a great influence on binding affinity which increased by almost an order of magnitude with an increase of the latter from 7 to 153 kDa. The binding affinity did not change significantly with DDA from 72 to 80% when the M(n) was kept constant near 80 kDa, but it increased substantially with DDA from 80 to 93% to reach a value similar to that obtained with chitosan of M(n) = 153 kDa and 80% DDA. These results provide insight into the previously reported dependence of the transfection efficiency of DNA/chitosan complexes on chitosan DDA and molecular weight, where complex stability and chitosan-DNA binding strength play a critical role.

Microcalorimetric study of adsorption of glycomacropeptide on anion-exchange chromatography adsorbent

The adsorption of glycomacropeptide (GMP) from cheese whey on an anion-exchange adsorbent was investigated using isothermal titration microcalorimetry to measure thermodynamic information regarding such processes. Isotherms data were measured at temperatures of 25 and 45 °C, pH 8.2 and various ionic strengths (0–0.08 mol L −1 NaCl). The equilibrium data were fit using the Langmuir model and the process was observed to be reversible. Temperature was observed to positively affect the interaction of the protein and adsorbent. Microcalorimetric studies indicated endothermic adsorption enthalpy in all cases, except at 45 °C and 0.0 mol L −1 NaCl. The adsorption process was observed to be entropically driven at all conditions studied. It was concluded that the increase in entropy, attributed to the release of hydration waters as well as bounded ions from the adsorbent and protein surface due to interactions of the protein and adsorbent, was a major driving force for the adsorption of GMP on the anion-exchange adsorbent. These results could allow for design of more effective ion-exchange separation processes for proteins.

Rigidity−Stability Relationship in Interlocked Model Complexes Containing Phenylene-Ethynylene-Based Disubstituted Naphthalene and Benzene

Structural rigidity has been found to be advantageous for molecules if they are to find applications in functioning molecular devices. In the search for an understanding of the relationship between the rigidity and complex stability in mechanically interlocked compounds, the binding abilities of two π-electron-rich model compounds (2 and 4), where rigidity is introduced in the form of phenylacetylene units, toward the π-electron deficient tetracationic cyclophane, cyclobis(paraquat-p-phenylene) (CBPQT^(4+)), were investigated. 1,4-Bis(2-(2-methoxyethoxy)ethoxy)-2,5-bis(2-phenylethynyl)benzene 2 and 1,5-bis(2-(2-methoxyethoxy)ethoxy)- 2,6-bis(2-phenylethynyl)naphthalene 4 were synthesized, respectively, from the appropriate precursor dibromides 1 and 3 of benzene and naphthalene carrying two methoxyethoxyethoxy side chains. The rigid nature of the compounds 2 and 4 is reflected in the reduced stabilities of their 1:1 complexes with CBPQT^(4+). Binding constants for both 2 (100 M^(-1)) and 4 (140 M^(-1)) toward CBPQT^(4+) were obtained by isothermal titration microcalorimetry (ITC) in MeCN at 25 °C. Compounds 1-4 were characterized in the solid state by X-ray crystallography. The stabilization within and beyond these molecules is achieved by a combination of intra- and intermolecular [C-H· · · O], [C-H· · ·π], and [π-π] stacking interactions. The diethyleneglycol chains present in compounds 1-4 are folded as a consequence of both intra- and intermolecular hydrogen bonds. The preorganized structures in both precursors 1 and 3 are repeated in both model compounds 2 and 4. In the structures of compounds 2 and 4, the geometry of the rigid backbone is differentsthe two terminal phenyl groups are twisted with respect to the central benzenoid ring in compound 2 and roughly perpendicular to the plane central naphthalene core in compound 4. To understand the significantly decreased stabilities of these complexes toward rigid guest molecules, relative to more flexible systems, we performed density functional theory (DFT) calculations using the newly developed M06-suite of density functionals. We conclude that the reduced binding abilities are a consequence of electronic and not steric factors, originating from the extended delocalization of the aromatic system.

Enthalpies and constants of dissociation of several neutral and cationic acids in aqueous and methanol/water solutions at various temperatures

The acidic dissociation enthalpies and constants of anilinium, protonated tris(hydroxymethyl)aminomethane (HTris +), benzoic and acetic acids, have been determined at several temperatures in pure water and in methanol/water mixtures by potentiometry and by isothermal titration microcalorimetry (ITC). The p K a values determined by both techniques are in accordance when the dissociation process involves large amounts of heat. However, for the neutral acids the ITC technique gave slightly lower p K a values than those from potentiometry at the highest temperatures studied due to the small amounts of heat involved in the acidic dissociation. The dissociation enthalpies have been determined directly by calorimetry and the obtained values slightly decrease with the increase of temperature. Therefore, only a rough estimation of the dissociation enthalpies can be obtained from potentiometric p K a by means of the Van’t Hoff approach.

Delineation of Alternative Conformational States in Escherichia coli Peptide Deformylase via Thermodynamic Studies for the Binding of Actinonin

We investigated the binding of a naturally occurring antibiotic, actinonin, to the Ni(2+)-reconstituted recombinant form of Escherichia coli peptide deformylase (PDF(Ec)) via isothermal titration microcalorimetry. The binding data conformed to both exothermic and endothermic phases with magnitudes of DeltaG degrees , DeltaH degrees , and TDeltaS degrees being equal to -12, -2.7, and 9.3 kcal/mol and -8.7, 3.9, and 12.6 kcal/mol, respectively. Evidently, although both phases are dominated by favorable entropic changes, the exothermic phase has about 6.7 kcal/mol enthalpic advantage over the endothermic phase. We observed that the removal of bound Ni(2+) from PDF(Ec) abolished the exothermic phase without affecting the endothermic phase, but it was regained upon addition of Zn(2+). In conjunction with metal analysis data, we propose that the recombinant form of PDF(Ec) is expressed in two stable conformational states that yield markedly distinct ITC profiles (i.e., exothermic versus endothermic) upon interaction with actinonin. The existence of two conformational states of PDF(Ec) is further supported by the observation of two distinct and independent transitions during the thermal unfolding of the enzyme. In addition, the thermodynamic data reveal that the formation of the PDF(Ec)-actinonin complex results in the transfer of one H(+) from the enzyme phase to the bulk solvent at pH 6.3. Both exothermic and endothermic phases produce highly negative DeltaC(p) degrees values, but there is no apparent enthalpy-entropy compensation effect upon formation of the PDF(Ec)-actinonin complex. In view of the known structural features of the enzyme, arguments are presented that the alternative conformational states of PDF(Ec) are modulated by the metal ligation at the enzyme site.

Direct Measurement Of Nonideal Mixing In Lipid Membranes

Lipid components in membranes are known to mix non-ideally, but the thermodynamics of this mixing remains poorly understood. Deviations from ideality may be characterized in part by the heat that is absorbed or released when components mix. This study aims to directly measure the heat of mixing of two phospholipid species in a bilayer membrane, using isothermal titration microcalorimetry. Unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phsophocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) were mixed in a calorimeter cell in the presence of methylated beta-cyclodextrin, which served as a lipid transfer catalyst. We expected on the basis of reduced head-group charge repulsions that mixing of the two lipids would be energetically favorable. The measured heat of mixing for a 1:1 mixture of POPC and POPG is -0.15 kJ/mol.

Comparative studies of interactions of hemoglobin with single-chain and with gemini surfactants

The interactions of hemoglobin with glutamic acid-based gemini surfactants (L 2G 2C n ) and with n-dodecylammonium α-glutamate (GDA) have been studied with isothermal titration microcalorimetry, fluorescence spectroscopy, UV–vis spectroscopy, and circular dichroism. The results indicate that GDA monomer can make the hemoglobin denatured, and that when the concentration of GDA is higher than cmc, heme monomer is released from the hydrophobic cavity of hemoglobin. On the other hand, L 2G 2C n surfactants can also interact with hemoglobin. Compared with GDA, L 2G 2C n have a much smaller binding ability with hemoglobin, and the circular dichroism spectra results show that the secondary structure of hemoglobin is possibly stabilized by a small amount of L 2G 2C n , which may generate hydrophobic linkages between the nonpolar residues of hemoglobin. However, with further addition of L 2G 2C n , the secondary structure of hemoglobin is unfolded, and the percentage of α helix in hemoglobin molecule is decreased. In addition, the L 2G 2C n surfactant with a longer spacer can reduce the denaturing degree of hemoglobin.

Effective embedding of liposomes into polyelectrolyte multilayered films: the relative importance of lipid-polyelectrolyte and interpolyelectrolyte interactions

The layer-by-layer (LbL) self-assembly of polyelectrolytes has emerged as a powerful and versatile strategy for engineering (bio)surfaces with active compounds. One possibility to engineer such films is to dope them with nanoparticles or with intact vesicles as carriers filled with the compounds of interest. In our previous studies we demonstrated that intact vesicles stabilized by poly-L-lysine (PLL) coating can be embedded in polyelectrolyte multilayer films made from the combination of either poly-L-glutamic acid (PGA) and poly(allylamine hydrochloride) (PAH) or from the combination of hyaluronic acid (HA) and PLL. These findings were empirical and not correlated to the interaction mode between PLL decorated vesicles (PLL-Lip) with the multilayer film. In order to produce robust and stable multi-layered nanocoatings containing intact vesicles, one has to understand how PLL-coated vesicles are integrated in the LbL architecture. To that aim, vesicle adsorption on three kinds of films, namely (PAH/PSS)12, where PSS stands for poly-(4-styrenesulfonate), (PAH/PGA)12, and (PLL/HA)12 was studied. PLL-Lip adsorption strongly depends on the interactions between PLL with both the vesicles and the polyanions used to form polyelectrolyte multilayers as was shown by direct evidence with confocal and atomic force microscopy as well as by the investigation of the interaction between PLL-Lip with the three polyanions in solution, namely HA, PGA and PSS. The latest was investigated by means of isothermal titration microcalorimetry. As a result, PSS in solution is able to desorb PLL from PLL coated liposomes, and on the other hand, PGA and HA can adsorb on PLL-stabilized vesicles without desorbing or inducing only partial desorption of the pre-adsorbed PLL. As a consequence, the (PLL/HA)12 film was found to be the best system among the three investigated combinations of polycations/polyanions for vesicle entrapment. The amount of entrapped vesicles is then proportional to the number of vesicle deposition steps and is increased for liposomes with higher charge. The release of a dye encapsulated in the entrapped vesicles could be induced by temperature changes which shows that for the HA-PLL combination of polyelectrolytes, the embedded vesicles retain their integrity below the main phase transition temperature. The temperature increase leads to fast leakage of liposome cargo while the film structure is not changed (up to 45 °C) suggesting that the lipidic bilayer is destabilized by ionic contacts with a polyelectrolyte network of the film. The latest is supported by the fact that within increase of vesicle negative charge, the stability of film-embedded vesicles is decreased whereas stability of these vesicles in solution is increased.

Enthalpies of Dilution of Colchicine in Aqueous NaCl Solutions

The concentration effect on the dilution enthalpies (ΔdilHm) of colchicine (COL) in aqueous NaCl solutions has been investigated by isothermal titration microcalorimetry at 298.15 K. The corresponding homogeneous enthalpic interaction coefficients have been calculated according to the excess enthalpy concept. The results show that the dilution enthalpies of COL in aqueous NaCl solutions at different mass fractions are positive. The overall trend is that enthalpies of dilution become more positive with the increase of the salt mass fraction. The values of enthalpic pair-wise interaction coefficients, h2, have been obtained by fitting the data of the enthalpies of dilution with a viral expansion. The results can be interpreted from the view of solute-solute and solute-solvent interactions involved in the solvent effects.

Isothermal Titration Calorimetry Reveals Differential Binding Thermodynamics of Variable Region-identical Antibodies Differing in Constant Region for a Univalent Ligand

The classical view of immunoglobulin molecules posits two functional domains defined by the variable (V) and constant (C) regions, which are responsible for antigen binding and antibody effector functions, respectively. These two domains are thought to function independently. However, several lines of evidence strongly suggest that C region domains can affect the specificity and affinity of an antibody for its antigen (Ag), independent of avidity-type effects. In this study, we used isothermal titration calorimetry to investigate the thermodynamic properties of the interactions of four V region-identical monoclonal antibodies with a univalent peptide antigen. Comparison of the binding of IgG1, IgG2a, IgG2b, and IgG3 with a 12-mer peptide mimetic of Cryptococcus neoformans polysaccharide revealed a stoichiometry of 1.9-2.0 with significant differences in thermodynamic binding parameters. Binding of this peptide to the antibodies was dominated by favorable entropy. The interaction of these antibodies with biotinylated peptides manifested greater enthalpy than for native peptides indicating that biotin labeling affected the types of Ag-Ab complexes formed. Our results provide unambiguous thermodynamic evidence for the notion that the C region can affect the interaction of the V region with an Ag.

Decompaction of Cationic Gemini Surfactant-Induced DNA Condensates by β-Cyclodextrin or Anionic Surfactant

Compaction of DNA by cationic gemini surfactant hexamethylene-1,6-bis-(dodecyldimethylammoniumbromide) (C12C6C12Br2) and the subsequent decompaction of the DNA-C12C6C12Br2 complexes by beta-cyclodextrin (beta-CD) or sodium dodecyl sulfate (SDS) have been studied by using zeta potential and particle size measurements, atomic force microscopy (AFM), isothermal titration microcalorimetry (ITC), and circular dichroism. The results show that C12C6C12Br2 can induce the collapse of DNA into densely packed bead-like structures with smaller size in an all-or-none manner, accompanied by the increase of zeta potential from highly negative values to highly positive values. In the decompaction of the DNA-C12C6C12Br2 complexes, beta-CD and SDS exhibit different behaviors. For beta-CD, the experimental results suggest that it can remove the outlayer hydrophobically bound C12C6C12Br2 molecules from the DNA-C12C6C12Br2 complexes by inclusion interaction, and the excess beta-CD may attach on the complexes by forming inclusion complexes with the hydrocarbon chains of the electrostatically bound C12C6C12Br2 that cannot be removed. The increase of steric hindrance due to the attachment of beta-CD molecules results in the decompaction of the DNA condensates though the true release of DNA cannot be attained. However, for SDS, the experimental results suggest that it can realize the decompaction and release of DNA from its complexes with C12C6C12Br2 due to both ion-pairing and hydrophobic interaction between SDS and C12C6C12Br2.

High Affinity Interaction between a Bivalve C-type Lectin and a Biantennary Complex-type N-Glycan Revealed by Crystallography and Microcalorimetry

Codakine is an abundant 14-kDa mannose-binding C-type lectin isolated from the gills of the sea bivalve Codakia orbicularis. Binding studies using inhibition of hemagglutination indicated specificity for mannose and fucose monosaccharides. Further experiments using a glycan array demonstrated, however, a very fine specificity for N-linked biantennary complex-type glycans. An unusually high affinity was measured by titration microcalorimetry performed with a biantennary Asn-linked nonasaccharide. The crystal structure of the native lectin at 1.3A resolution revealed a new type of disulfide-bridged homodimer. Each monomer displays three intramolecular disulfide bridges and contains only one calcium ion located in the canonical binding site that is occupied by a glycerol molecule. The structure of the complex between Asn-linked nonasaccharide and codakine has been solved at 1.7A resolution. All residues could be located in the electron density map, except for the capping beta1-4-linked galactosides. The alpha1-6-linked mannose binds to calcium by coordinating the O3 and O4 hydroxyl groups. The GlcNAc moiety of the alpha1,6 arm engages in several hydrogen bonds with the protein, whereas the GlcNAc on the other antenna is stacked against Trp(108), forming an extended binding site. This is the first structural report for a bivalve lectin.

Sequence specific and high affinity recognition of 5′-ACGCGT-3′ by rationally designed pyrrole-imidazole H-pin polyamides: Thermodynamic and structural studies

Imidazole (Im) and Pyrrole (Py)-containing polyamides that can form stacked dimers can be programmed to target specific sequences in the minor groove of DNA and control gene expression. Even though various designs of polyamides have been thoroughly investigated for DNA sequence recognition, the use of H-pin polyamides (covalently cross-linked polyamides) has not received as much attention. Therefore, experiments were designed to systematically investigate the DNA recognition properties of two symmetrical H-pin polyamides composed of PyImPyIm ( 5) or f-ImPyIm ( 3e, f = formamido) tethered with an ethylene glycol linker. These compounds were created to recognize the cognate 5′-ACGCGT-3′ through an overlapped and staggered binding motif, respectively. Results from DNaseI footprinting, thermal denaturation, circular dichroism, surface plasmon resonance and isothermal titration microcalorimetry studies demonstrated that both H-pin polyamides bound with higher affinity than their respective monomers. The binding affinity of formamido-containing H-pin 3e was more than a hundred times greater than that for the tetraamide H-pin 5, demonstrating the importance of having a formamido group and the staggered motif in enhancing affinity. However, compared to H-pin 3e, tetraamide H-pin 5 demonstrated superior binding preference for the cognate sequence over its non-cognates, ACCGGT and AAATTT. Data from SPR experiments yielded binding constants of 1.6 × 10 8 M −1 and 2.0 × 10 10 M −1 for PyImPyIm H-pin 5 and f-ImPyIm H-pin 3e, respectively. Both H-pins bound with significantly higher affinity (ca. 100-fold) than their corresponding unlinked PyImPyIm 4 and f-ImPyIm 2 counterparts. ITC analyses revealed modest enthalpies of reactions at 298 K (Δ H of −3.3 and −1.0 kcal mol −1 for 5 and 3e, respectively), indicating these were entropic-driven interactions. The heat capacities (Δ C p) were determined to be −116 and −499 cal mol −1 K −1, respectively. These results are in general agreement with Δ C p values determined from changes in the solvent accessible surface areas using complexes of the H-pins bound to (5′-CC ACGCGTGG) 2. According to the models, the H-pins fit snugly in the minor groove and the linker comfortably holds both polyamide portions in place, with the oxygen atoms pointing into the solvent. In summary, the H-pin polyamide provides an important molecular design motif for the discovery of future generations of programmable small molecules capable of binding to target DNA sequences with high affinity and selectivity.

Zn2+‐linked dimerization of UreG from Helicobacter pylori, a chaperone involved in nickel trafficking and urease activation

The biosynthesis of the active metal-bound form of the nickel-dependent enzyme urease involves the formation of a lysine-carbamate functional group concomitantly with the delivery of two Ni(2+) ions into the precast active site of the apoenzyme and with GTP hydrolysis. In the urease system, this role is performed by UreG, an accessory protein belonging to the group of homologous P-loop GTPases, often required to complete the biosynthesis of nickel-enzymes. This study is focused on UreG from Helicobacter pylori (HpUreG), a bacterium responsible for gastric ulcers and cancer, infecting large part of the human population, and for which urease is a fundamental virulence factor. The soluble HpUreG was expressed in E. coli and purified to homogeneity. On-line size exclusion chromatography and light scattering indicated that apo-HpUreG exists as a monomer in solution. Circular dichroism, which demonstrated the presence of a well-defined secondary structure, and NMR spectroscopy, which revealed a large number of residues that appear structured on the basis of their backbone amide proton chemical shift dispersion, indicated that, at variance with other UreG proteins so far characterized, this protein is significantly folded in solution. The amino acid sequence of HpUreG is 29% identical to that of HypB from Methanocaldococcus jannaschii, a dimeric zinc-binding GTPase involved in the in vivo assembly of [Ni,Fe]-hydrogenase. A homology-based molecular model of HpUreG was calculated, which allowed us to identify structural and functional features of the protein. Isothermal titration microcalorimetry demonstrated that HpUreG specifically binds 0.5 equivalents of Zn(2+) per monomer (K(d) = 0.33 +/- 0.03 microM), whereas it has 20-fold lower affinity for Ni(2+) (K(d) = 10 +/- 1 microM). Zinc ion binding (but not Ni(2+) binding) causes protein dimerization, as confirmed using light scattering measurements. The structural rearrangement occurring upon Zn(2+)-binding and consequent dimerization was evaluated using circular dichroism and fluorescence spectroscopy. Fully conserved histidine and cysteine residues were identified and their role in zinc binding was verified by site-directed mutagenesis and microcalorimetry. The results are analyzed and discussed with respect to analogous examples of GTPases in nickel metabolism.

Two‐Phase Calorimetry. I. Studies on the Thermodynamics of Lanthanide Extraction by Bis(2‐EthylHexyl) Phosphoric Acid

Enthalpies of extraction of lanthanide ions by bis(2‐ethylhexyl) phosphoric acid (HDEHP) from aqueous nitrate solutions have been determined using isothermal titration microcalorimetry. This solvent extraction system was chosen as a model for developing a direct method of calorimetric investigation of thermochemistry of liquid‐liquid distribution reactions because it has been well‐studied and is predictable. To provide a quantitative validation for a two‐phase calorimetric technique, the enthalpy of extraction of europium(III) by HDEHP also was determined applying the more conventional method of van't Hoff analysis of temperature dependent extraction equilibrium constants. Within the accuracy of the measurements, the determination of thermodynamic parameters by the complementary techniques agreed. The extraction enthalpies determined in this study have been correlated with reported free energies of extraction of the lanthanides to reveal the patterns of extraction entropies observed across the lanthanide series. Challenges and opportunities for application of two‐phase calorimetry as a new analytical method for routine analyses of the thermochemistry of solvent extraction reactions are discussed.

Heterocyclic diamidine interactions at AT base pairs in the DNA minor groove: effects of heterocycle differences, DNA AT sequence and length.

Given the increasing significance of diamidines as DNA-targeted therapeutics and biotechnology reagents, it is important to establish the variations in thermodynamic quantities that characterize the interactions of closely related compounds to different sequence AT binding sites. In this study, an array of methods including biosensor-surface plasmon resonance (SPR), isothermal titration microcalorimetry (ITC), circular dichroism (CD), thermal melting (Tm) and molecular modeling have been used to characterize the binding of dicationic diamidines related to DB75 (amidine-phenyl-furan-phenyl-amidine) with alternating and nonalternating AT sequences. Conversion of the central furan of DB75 to other similar groups, such as thiophene or selenophene, can yield compounds with increased affinity and sequence binding selectivity for the minor groove. Calorimetric measurements revealed that the thermodynamic parameters (Delta G, Delta H, Delta S) that drive diamidine binding to alternating and nonalternating oligomers can be quite different and depend on both DNA sequence and length. Small changes in a compound can have major effects on DNA interactions. By choosing an appropriate central group it is possible to "tune" the shape of the molecule to match DNA for enhanced affinity and sequence recognition.