Interactions In Complex Formation Research Articles

Fabrication and characterization of lysozyme fibrils/Zein complexes for resveratrol encapsulation: Improving stability, antioxidant and antibacterial activities

Resveratrol (Res), a naturally occurring hydrophobic polyphenol, boasts numerous health-promoting bio-functionalities. However, its limited water solubility and stability impede further applications in the food industry. This study aims to address these challenges by fabricating stable Res-loaded lysozyme fibrils/zein (Ly-F/Z) complexes. The complexes were prepared using an antisolvent precipitation method. The interaction mechanism between Ly-F and zein was elucidated through dynamic light scattering, Fourier-transform infrared spectroscopy and dissociative experiments, revealing the involvement of hydrogen bonding, electrostatic forces and hydrophobic interactions in complex formation. The Ly-F/Z complexes were utilized to encapsulate Res, resulting in an encapsulation efficiency of 82.58 %. X-ray diffraction analysis confirmed the successful encapsulation of Res within Ly-F/Z complexes, presenting an amorphous state. The Ly-F/Z-Res complexes exhibited a “fruit tree” morphology with dense fruit, showcasing remarkable stability, antioxidant and antibacterial activities. Consequently, the Ly-F/Z complexes can serve as promising delivery systems for Res in functional foods.

Exploration of diverse interactions of nonionic poly(vinylpyrrolidone) (PVP) and anionic sodium carboxymethylcellulose (NaCMC) polymers in aqueous imidazolium-based surface active ionic liquid solutions

The binding interactions in complex formation between surface active ionic liquids (SAILs) and polymers (nonionic or anionic) are gaining attention because of their potential applications in novel materials. Therefore, the use of a variety of measurements is required for a better understanding of the interactions involved in polymer/SAIL formation. Here, we have investigated the interactions of nonionic poly(vinylpyrrolidone) (PVP) and anionic sodium carboxymethylcellulose (NaCMC) polymers with SAIL 1-tetradecyl-3-methylimidazolium chloride [C14mim][Cl] in aqueous solution using different measurements such as conductivity, surface tension, fluorescence spectroscopy, dynamic light scattering (DLS) and rheometric measurements. The surface tension, conductivity and fluorescence profiles for SAIL-NaCMC systems provide distinct breakpoints that correspond to four different aggregation states: the concentration at which SAIL monomers begin to attach to the polymer chain (C1), the critical aggregation concentration C2 (cac), the saturation concentration (CS) and the critical micelle concentration (cmc). In the case of SAIL-PVP systems, the surface tension and fluorescence profiles confer three transition states: C2 (cac), CS and cmc and only two transition states have been observed in conductivity curves. The cooperative binding of SAIL to the NaCMC/PVP chain at the solution interface was analyzed by calculating various thermodynamic and interfacial parameters. Strong complexation was observed between NaCMC-SAIL molecules compared to PVP-SAIL systems at the air-solution surface. This is due to the non-ionic character of the PVP chain, which shows weak interactions with SAIL in comparison to NaCMC. Furthermore, DLS and fluorescence measurements were used to investigate the interactions between SAIL and polymers.

Interplay of Drug-Polymer Interactions and Release Performance for HPMCAS-Based Amorphous Solid Dispersions.

The interplay between drug and polymer chemistry and its impact on drug release from an amorphous solid dispersion (ASD) is a relatively underexplored area. Herein, the release rates of several drugs of diverse chemistry from hydroxypropyl methylcellulose acetate succinate (HPMCAS)-based ASDs were explored using surface area normalized dissolution. The tendency of the drug to form an insoluble complex with HPMCAS was determined through coprecipitation experiments. The role of pH and the extent of drug ionization were probed to evaluate the role of electrostatic interactions in complex formation. Relationships between the extent of complexation and the drug release rate from an ASD were observed, whereby the drugs could be divided into two groups. Drugs with a low extent of insoluble complex formation with HPMCAS tended to be neutral or anionic and showed reasonable release at pH 6.8 even at higher drug loadings. Cationic drugs formed insoluble complexes with HPMCAS and showed poor release when formulated as an ASD. Thus, and somewhat counterintuitively, a weakly basic drug showed a reduced release rate from an ASD at a bulk solution pH where it was ionized, relative to when unionized. The opposite trend was observed in the absence of polymer for the neat amorphous drug. In conclusion, electrostatic interactions between HPMCAS and lipophilic cationic drugs led to insoluble complex formation, which in turn resulted in ASDs with poor release performance.

Self‐Assembly of Lipoaminoacids‐DNA Based on Thermodynamic and Aggregation Properties

AbstractLipoamino acids (LAA) are biocompatible and biodegradable biosurfactants, a promising alternative to viral vectors in gene delivery. LAA are constituted by a polar head, the amino acid, and a hydrocarbon (alkyl) chain usually from a fatty acid or fatty acid derivative, such as a fatty amine or a fatty alcohol. In this work, dodecyl LAA was produced from dodecylamine and natural l‐amino acids cystine (Cys), lysine (Lys), and phenylalanine (Phe) using an enzyme‐based approach with porcine pancreatic lipase. The self‐assembly behavior of LAA solutions, in the absence or presence of DNA, was studied by conductivity and fluorescence regarding the application as transfection agents. Conductivity measurements yielded important system parameters, including critical micelle concentration (CMC) and standard Gibbs energy of micellization (ΔG°mic) for pure LAA systems, and apparent critical aggregation concentration (CACapp) and apparent standard Gibbs energy of aggregation (ΔG°agg) for the mixed LAA‐DNA systems. The CMC increased in the order of decreasing lipophilicity: (C12Cys)2 < C12Phe < C12Lys, CMC values were higher in the presence of DNA, suggesting the formation of a LAA‐DNA complex responsible for hindering the micellization process. Binding of the LAA with DNA was confirmed from fluorescence measurements for the ethidium bromide exclusion assay. Results suggest a weak interaction of the LAA with DNA which can be attributed to their relatively short dodecyl chains and/or the ionic strength of the buffer solution, supporting the role of hydrophobic interactions in complex formation between DNA and the oppositely charged surfactant in combination with electrostatic interactions. The CACapp values decreased with increasing LAA hydrophobicity, reflecting the relevance of hydrophobic interactions in complex coacervation.

All That Binds Is Not Gold-The Relative Weight of Aurophilic Interactions in Complex Formation.

Often have you heard that complexes containing close Au(I) contacts are strongly influenced by what has come to be known as aurophilic interactions. In this work, local orbital analysis is carried out to separate competing metallophilic and other weak interactions at the correlated level in three selected molecular crystals. We carefully separate and discuss the different contributions to the total interaction energy of dimers and trimers according to their spatial location, and identify the relative weight in binding. Changes according to the orientation of the monomers have also been computed. Our results show that although metallophilic contacts contribute to the overall stability and the structure of the crystals, they are not at all dominant, and ligand-ligand interactions can easily outweigh the latter.

Comparative investigation of interactions between two ruthenium(II) arene PTA type complexes with curcuminoid ligands and human serum albumin

Ruthenium(II) arene PTA (1,3,5-triaza-7-phosphaadamantane) type complexes with curcuminoid ligands have attracted great attentions in biomedical areas. Investigation of structural influences helps in understanding the biological effects of these complexes. To research their structural influences, the interactions between two ruthenium(II) arene PTA type complexes with curcuminoid ligands and human serum albumin (HSA) were systematically investigated by multispectroscopic techniques and electrochemical methods. The fluorescence spectral results indicated that two complexes bonded with Sudlow's site I of HSA to form 1: 1 complex−HSA compounds. Van der Waals interactions and hydrogen bonds formation were the major binding forces during their complex formation interactions. Subsequently, the intrinsic fluorescence of HSA was statically quenched by these complexes through concentration-dependent manner. The conformation and secondary structure of HSA were all changed at the present of two complexes. The space steric hindrance of complexes was responsible for differences in the fluorescence quenching, while the chemical polarity played important role in the variation of binding interactions between HSA and two complexes. These results provide the molecular understanding of binding interactions between protein and ruthenium(II) arene PTA type complexes with curcuminoid ligands, which gain new insight into the biological applications of similar ruthenium(II) arene derivatives in the future.

Роль электростатических взаимодействий в формировании комплекса между бактериальной люциферазой и NADPH:FMN-оксидоредуктазой

Роль электростатических взаимодействий в формировании комплекса между бактериальной люциферазой и NADPH:FMN-оксидоредуктазой

Structures of the nucleoid occlusion protein SlmA bound to DNA and the C-terminal domain of the cytoskeletal protein FtsZ

Cell division in most prokaryotes is mediated by FtsZ, which polymerizes to create the cytokinetic Z ring. Multiple FtsZ-binding proteins regulate FtsZ polymerization to ensure the proper spatiotemporal formation of the Z ring at the division site. The DNA-binding protein SlmA binds to FtsZ and prevents Z-ring formation through the nucleoid in a process called "nucleoid occlusion" (NO). As do most FtsZ-accessory proteins, SlmA interacts with the conserved C-terminal domain (CTD) that is connected to the FtsZ core by a long, flexible linker. However, SlmA is distinct from other regulatory factors in that it must be DNA-bound to interact with the FtsZ CTD. Few structures of FtsZ regulator-CTD complexes are available, but all reveal the CTD bound as a helix. To deduce the molecular basis for the unique SlmA-DNA-FtsZ CTD regulatory interaction and provide insight into FtsZ-regulator protein complex formation, we determined structures of Escherichia coli, Vibrio cholera, and Klebsiella pneumonia SlmA-DNA-FtsZ CTD ternary complexes. Strikingly, the FtsZ CTD does not interact with SlmA as a helix but binds as an extended conformation in a narrow, surface-exposed pocket formed only in the DNA-bound state of SlmA and located at the junction between the DNA-binding and C-terminal dimer domains. Binding studies are consistent with the structure and underscore key interactions in complex formation. Combined, these data reveal the molecular basis for the SlmA-DNA-FtsZ interaction with implications for SlmA's NO function and underscore the ability of the FtsZ CTD to adopt a wide range of conformations, explaining its ability to bind diverse regulatory proteins.

Inclusion complexes of HP-β-cyclodextrin with agomelatine: Preparation, characterization, mechanism study and in vivo evaluation

Agomelatine (AGM), is efficacious in both the acute phase and the continuation phase of depression. However, its poor water-solubility, low bioavailability and polymorphism limit its pharmacological effects. To address these problems, agomelatine-hydroxypropyl-β-cyclodextrin inclusion complex (AGM/HPβ-CD) was prepared successfully by freeze-drying. The products was evaluated by structural characterization, solubilization test, in-situ absorption of rat intestinal tract and pharmacokinetic study. In addition, thermodynamic studies were performed, the results indicated that the inclusion process was enthalpy-determined and exothermic nature of complexation, signifying the role of steric interactions in complex formation. Molecular docking of AGM with HPβ-CD has been conducted as well to verify the experimental findings and predict the stable molecular structure of the inclusion complex. The in vivo data showed that, AGM was mainly absorbed in duodenum and jejunum by passive diffusion. AGM/HPβ-CD inclusion complex displayed earlier Tmax and higher Cmax, and the AUC0-12h was approximately twice larger than its physical mixture. These results suggested that AGM/HPβ-CD inclusion complex was established with 1:1 stoichiometry through the naphthalene group of AGM and it was deeply inserted into the cavity of HPβ-CD, and the inclusion complex could significantly enhance the oral bioavailability of AGM.

Nano-amorphous composites of cilostazol–HP-β-CD inclusion complexes: physicochemical characterization, structure elucidation, thermodynamic studies and in vitro evaluation

The present research work aims to obtain nano-amorphous composites of cilostazol and HP-β-CD using combinatorial spray drying and inclusion complexation technique. Physicochemical characterization of complexes was performed in solution and solid state to understand the mode of inclusion of cilostazol in the host cavity. Phase solubility studies revealed formation of first-order soluble complex with stability constant of 892.13 M−1, while Job’s plot confirmed 2:1 (HP-β-CD:CLZ) stoichiometry. Structure elucidation of the complex was performed using UV, FTIR and NMR spectroscopy. 1H NMR spectra revealed only marginal chemical shifts for both CLZ and HP-β-CD suggesting shallow penetration and presence of van der Waals forces. Proximity relationships between CLZ and HP-β-CD obtained from 2D (ROESY) NMR assisted computer simulations to compute the binding energy of this complex. Circular dichroism gave an estimate of the orientation of CLZ in the HP-β-CD cavity. Solid state complexes were evaluated using FTIR, DSC and hot stage microscopy, all of which confirmed inclusion complexation. The amorphous nature of the complex was established using XRD and atomic force microscopy. Thermodynamic studies indicate exothermic nature of complexation while signifying the role of steric interactions in complex formation. The spray dried amorphous nanocomposites exhibited lyotropic phase transitions with varying relative humidity as suggested by dynamic vapor sorption analysis. In vitro biorelevant dissolution media studies exhibited negligible food effects as indicated by the fed/fasted ratio.

Competitive reactions in solutions of the complex of chitosan and DNA

The interaction of water-soluble modified chitosan with a polymethacrylate anion and DNA in aqueous and water-salt solutions is studied via the methods of fluorescence quenching with the use of a pyrenyl-labeled polyacid and the intercalated dye ethidium bromide. After passage from neutral to weakly acidic solutions, the stability of the polysaccharide complex with the polymethacrylate anion significantly increases, but, in the case of the DNA-containing complex, stability remains almost invariable. In spite of a much higher stability of complexes against salt in weakly ionized poly(methacrylic acid), the equilibrium of the competitive binding of chitosan shifts toward complexation with DNA, a result that is indicative of the dominant role of electrostatic interactions in complex formation. The capability for sharp and reversible variation in the state of the system in weakly acidic media may become the starting point for development of biotechnological systems for separation of biological mixtures and creation of vectors for delivery of genetic material that are based on cationic biodegradable polysaccharides.

Cation−π Interaction in Complex Formation Between Tl+ Ion and Calix[4]crown-6 and Some Calix[4]biscrown-6 Derivatives: Thallium-203 NMR, Proton NMR, and X-ray Evidence

Metallocapped complexation of the thallium(I) ion with calix[4]crown-6 (1) and three different calix[4]biscrown-6 derivatives (2-4) has been investigated by a combination of (203)Tl and (1)H NMR in a CD(3)CN/CDCl(3) (4:1 v/v) solution. The results clearly revealed the formation in solution of mono- and dithallium(I) complexes in which the metal cations are held in the ligands' cavities close to the calix[4]arene ring. In addition, a solid state mononuclear complex between 1,3-calix[4]bis-o-benzo-crown-6 (3) and TlClO(4) was prepared, and its X-ray crystal structure was determined. The results of the structural data in solution and the solid state suggested that the calix[4]crown-6 derivatives considered provide pi cavities as active sites for the complexation of thallium(I) ions.

Expression of outer mitochondrial membrane cytochrome b 5 in Escherichia coli. Purification of the recombinant protein and studies of its interaction with electron-transfer partners

In the present work, we report expression in Escherichia coli, purification, and characterization of recombinant full-length cytochrome b(5) from outer mitochondrial membrane. Optimization of expression conditions for cytochrome b(5) from outer mitochondrial membrane allowed reaching expression level up to 10(4) nmol of the hemeprotein per liter of culture. Recombinant cytochrome b(5) from outer mitochondrial membrane was purified from cell lysate by using metal-affinity chromatography. It has physicochemical, spectral, and immunochemical properties similar to those of cytochrome b(5) from rat liver outer mitochondrial membrane. Immobilized recombinant mitochondrial cytochrome b(5) was used as affinity ligand to study its interaction with electron transfer proteins. By using this approach, it is shown that in interaction of NADPH:cytochrome P450 reductase with both forms of cytochrome b(5) an important role is played by hydrophobic interactions between proteins, although the contribution of these interactions in complex formation with NADPH:cytochrome P450 reductase is different for isoforms of cytochrome b(5).

Salt Enhances Calmodulin-Target Interaction

Calmodulin (CaM) operates as a Ca 2+ sensor and is known to interact with and regulate hundreds of proteins involved in a great many aspects of cellular function. It is of considerable interest to understand the balance of forces in complex formation of CaM with its target proteins. Here we have studied the importance of electrostatic interactions in the complex between CaM and a peptide derived from smooth-muscle myosin light-chain kinase by experimental methods and Monte Carlo simulations of electrostatic interactions. We show by Monte Carlo simulations that, in agreement with experimental data, the binding affinity between CaM and highly charged peptides is surprisingly insensitive to changes in the net charge of both the protein and peptide. We observe an increase in the binding affinity between oppositely charged partners with increasing salt concentration from zero to 100 mM, showing that formation of globular CaM-kinase type complexes is facilitated at physiological ionic strength. We conclude that ionic interactions in complex formation are optimized at pH and saline similar to the cell environment, which probably overrules the electrostatic repulsion between the negatively charged Ca 2+-binding domains of CaM. We propose a conceivable rationalization of CaM electrostatics associated with interdomain repulsion.

Electron donation between copper containing nitrite reductases and cupredoxins: the nature of protein-protein interaction in complex formation

In denitrifying organisms with copper containing dissimilatory nitrite reductases, electron donation from a reduced cupredoxin is an essential step in the reduction of nitrite to nitric oxide. Copper nitrite reductases are categorised into two subgroups based on their colour, green and blue, which are found in organisms where the cupredoxins are pseudoazurins and azurins, respectively. In view of this and some in vitro electron donation experiments, it has been suggested that copper nitrite reductases have specific electron donors and that electron transfer takes place in a specific complex of the two proteins. We report results from the first comprehensive electron donation experiments using three copper nitrite reductases, one green and two blue, and five cupredoxins, one pseudoazurin and four azurins. Our data show that pseudoazurin can readily donate electrons to both blue and green copper nitrite reductases. In contrast, all of the azurins react very sluggishly as electron donors to the green nitrite reductase. These results are discussed in terms of surface compatibility of the component proteins, complex formation, overall charges, charge distribution, hydrophobic patches and redox potentials. A docking model for the complexes is proposed.

Mechanism of NADPH oxidase activation by the Rac/Rho-GDI complex.

The low molecular weight GTP binding protein Rac is essential to the activation of the NADPH oxidase complex, involved in pathogen killing during phagocytosis. In resting cells, Rac exists as a heterodimeric complex with Rho GDP dissociation inhibitor (Rho-GDI). Two types of interactions exist between Rac and Rho-GDI: a protein-lipid interaction, implicating the polyisoprene of the GTPase, as well as protein-protein interactions. Using the two-hybrid system, we show that nonprenylated Rac1 interacts very weakly with Rho-GDI, pointing to the predominant role of protein-isoprene interaction in complex formation. In the absence of this strong interaction, we demonstrate that three sites of protein-protein interaction, Arg66(Rac)-Leu67(Rac), His103(Rac), and the C-terminal polybasic region Arg183(Rac)-Lys188(Rac), are involved and cooperate in complex formation. When Rac1 mutants are prenylated by expression in insect cells, they all interact with Rho-GDI. Rho-GDI is able to exert an inhibitory effect on the GDP/GTP exchange reaction except in the complex in which Rac1 has a deletion of the polybasic region (Arg183(Rac)-Lys188(Rac)). This complex is, most likely, held together through protein-lipid interaction only. Although able to function as GTPases, the mutants of Rac1 that failed to interact with Rho-GDI also failed to activate the NADPH oxidase in a cell-free assay after loading with GTP. Mutant Leu119(Rac)Gln could both interact with Rho-GDI and activate the NADPH oxidase. The Rac1/Rho-GDI and Rac1(Leu119Gln)/Rho-GDI complexes, in which the GTPases were bound to GDP, were found to activate the oxidase efficiently. These data suggest that Rho-GDI stabilizes Rac in an active conformation, even in the GDP-bound state, and presents it to its effector, the p67phox component of the NADPH oxidase.

The heterodimeric complex of MRP-8 (S100A8) and MRP-14 (S100A9). Antibody recognition, epitope definition and the implications for structure.

The S100 calcium-binding proteins MRP-8 (S100A8) and MRP-14 (S100A9) form a heterodimeric complex in the cytosol of monocyte and neutrophil cell types circulating in peripheral blood. This complex, but not the individual subunit proteins, is specifically recognized by mAb 27E10. Domains in MRP-8 and MRP-14 mediating heterodimeric complex formation have not yet been identified but it is predicted that the structure of the complex will be similar to homodimeric forms of other S100 proteins. This study makes use of the specificity of mAb 27E10, and an in vitro coupled transcription/translation system to further examine the formation and maintenance of the MRP-8/MRP-14 complex. Truncated mutants of MRP-14 that lack the N-terminal residues 1-4 or the extended C-terminal 'tail', both complex with MRP-8. These deleted domains of MRP-14 are therefore not essential for complex formation. Peptides from MRP-8 or MRP-14, used to induce the epitope recognized by mAb 27E10, show that a critical interaction in complex formation involves the N-terminal of MRP-8 interacting with MRP-14. Phage display analysis defined composite residues of the epitope recognized by mAb 27E10. The epitope is trans-subunit, composed of residues in the C-terminal ends of helix IV in MRP-14 and helix I of MRP-8. A further complex-specific mAb, named 5.5, recognizes the hydrophobic residues in helix IV of MRP-8, exposed during heterodimer formation. The definition of these two epitopes indicates that helices IV of MRP-8 and MRP-14 are also a prominent point of interaction and suggests that the subunit proteins will assume an antiparallel alignment in the heterodimer, similar in structure to the homodimeric forms of S100 proteins.

Electron transfer reactions in both the oxidizing and reducing sites of photosystem I: A laser flash absorption spectroscopy study

The rate constants of electron transfer from reduced plastocyanin and cytochrome c-552, obtained from the green alga Monoraphidium braunii, to the photo-oxidized P700 (P700+) in photosystem I (PSI) particles from spinach were determined using the laser flash absorption spectroscopy technique. The redox properties of both proteins were found to be quite similar, which is consistent with their physiological role of substituting for one another as electron donors to PSI. In analogous experiments, the electron transfer reactions from the reducing site of PSI to oxidized ferredoxin, isolated from both spinach and Monoraphidium braunii, were studied. Second-order rate constants of PSI reduction by either cytochrome c-552 or plastocyanin and oxidation by ferredoxin are of the same order of magnitude (approximately 107 M−1 s−1), whereas the corresponding first-order rate constants are significantly greater (approximately 102) for PSI reduction than for its oxidation. In all cases, the protein concentration dependencies of the reaction rates are characteristic of a mechanism involving the formation of a transient complex preceding electron transfer, whereas the effects of salt ions and pH suggest the occurrence of attractive electrostatic interactions in complex formation between the redox metalloproteins and their respective binding sites in PSI. Divalent cations such as magnesium appear to play a specific role in the interaction between PSI and both the donor and acceptor proteins.

Electron transfer reactions in both the oxidizing and reducing sites of photosystem I: A laser flash absorption spectroscopy study

The rate constants of electron transfer from reduced plastocyanin and cytochrome c-552, obtained from the green alga Monoraphidium braunii, to the photo-oxidized P700 (P700 +) in photosystem I (PSI) particles from spinach were determined using the laser flash absorption spectroscopy technique. The redox properties of both proteins were found to be quite similar, which is consistent with their physiological role of substituting for one another as electron donors to PSI. In analogous experiments, the electron transfer reactions from the reducing site of PSI to oxidized ferredoxin, isolated from both spinach and Monoraphidium braunii, were studied. Second-order rate constants of PSI reduction by either cytochrome c-552 or plastocyanin and oxidation by ferredoxin are of the same order of magnitude (approximately 10 7 M −1 s −1), whereas the corresponding first-order rate constants are significantly greater (approximately 10 2) for PSI reduction than for its oxidation. In all cases, the protein concentration dependencies of the reaction rates are characteristic of a mechanism involving the formation of a transient complex preceding electron transfer, whereas the effects of salt ions and pH suggest the occurrence of attractive electrostatic interactions in complex formation between the redox metalloproteins and their respective binding sites in PSI. Divalent cations such as magnesium appear to play a specific role in the interaction between PSI and both the donor and acceptor proteins.

Thermodynamic analysis of the lactose repressor-operator DNA interaction.

Kinetic and equilibrium constants for lactose repressor-operator DNA interaction have been examined as a function of salt concentration, size and sequence context of the operator DNA, and temperature. Significant salt effects were observed on kinetic and equilibrium parameters for pLA 322-8, an operator-containing derivative of pBR 322, and pIQ, an operator and pseudooperator-containing derivative of pBR 322. The association rate constant and equilibrium constant for the 40 base pair operator fragment were also salt dependent. Data for all the DNAs were consistent with a sliding mechanism for repressor-operator association/dissociation [Berg, O. G., & Blomberg, C. (1978) Biophys. Chem. 8, 271-280]. Calculation of the number of ionic interactions based on salt dependence yielded a value of approximately 8 for repressor binding to pIQ and pLA 322-8 vs. approximately 6 for the repressor-40 base pair fragment. These data and the differences in binding parameters for the plasmids vs. the 40 base pair operator are consistent with the formation of an intramolecular ternary complex in the plasmid DNAs. Unusual biphasic temperature dependence was observed in the equilibrium and dissociation rate constants for pLA 322-8, pIQ, and the 40 base pair fragment. These observations coupled with a discontinuity found in the inducer association rate constant as a function of temperature suggest a structural change in the protein. The large positive entropy contributions associated with repressor binding to all the DNAs examined provide the significant driving force for the reaction and are consistent with involvement of ionic and apolar interactions in complex formation.