Cooperative Binding Model Research Articles

Reaction of Cd(II)–Morin with dsDNA for biosensing of ssDNA oligomers with complementary, GCE-immobilized ssDNA

In this work, the complex cadmium(II)–morin was synthesized and its interaction with double-stranded salmon sperm DNA was studied by electrochemical methods on glassy carbon electrode (GCE). It was shown that Cd(II)–Morin with high electrochemical activity can intercalate into the double-helix DNA, and the binding stoichiometry and equilibrium dissociation constant according to the Hill model for cooperative binding were calculated to be 1.761 and 2.5 × 10 − 5 M, respectively. Using Cd(II)–Morin as a novel hybridization indicator, the hybridization between the probe and its complementary and mismatched sequence was investigated by differential pulse voltammetry (DPV), which was to access the selectivity of the developed electrochemical DNA biosensor. The complementary target ssDNA could be quantified over the range from 2.69 × 10 − 8 M to 9.16 × 10 − 7 M with a linear correlation of 0.9971 and a detection limit of 9.30 × 10 − 9 M. These results demonstrated that the Cd(II)–Morin indicator provides great promise for the rapid and selective measurement of the target DNA.

Detecting cis -regulatory binding sites for cooperatively binding proteins

Several methods are available to predict cis-regulatory modules in DNA based on position weight matrices. However, the performance of these methods generally depends on a number of additional parameters that cannot be derived from sequences and are difficult to estimate because they have no physical meaning. As the best way to detect cis-regulatory modules is the way in which the proteins recognize them, we developed a new scoring method that utilizes the underlying physical binding model. This method requires no additional parameter to account for multiple binding sites; and the only necessary parameters to model homotypic cooperative interactions are the distances between adjacent protein binding sites in basepairs, and the corresponding cooperative binding constants. The heterotypic cooperative binding model requires one more parameter per cooperatively binding protein, which is the concentration multiplied by the partition function of this protein. In a case study on the bacterial ferric uptake regulator, we show that our scoring method for homotypic cooperatively binding proteins significantly outperforms other PWM-based methods where biophysical cooperativity is not taken into account.

Crystal structure of the λ repressor and a model for pairwise cooperative operator binding

Bacteriophage lambda has for many years been a model system for understanding mechanisms of gene regulation. A 'genetic switch' enables the phage to transition from lysogenic growth to lytic development when triggered by specific environmental conditions. The key component of the switch is the cI repressor, which binds to two sets of three operator sites on the lambda chromosome that are separated by about 2,400 base pairs (bp). A hallmark of the lambda system is the pairwise cooperativity of repressor binding. In the absence of detailed structural information, it has been difficult to understand fully how repressor molecules establish the cooperativity complex. Here we present the X-ray crystal structure of the intact lambda cI repressor dimer bound to a DNA operator site. The structure of the repressor, determined by multiple isomorphous replacement methods, reveals an unusual overall architecture that allows it to adopt a conformation that appears to facilitate pairwise cooperative binding to adjacent operator sites.

Cooperative Binding of the Class I Major Histocompatibility Complex Cytoplasmic Domain and Human Immunodeficiency Virus Type 1 Nef to the Endosomal AP-1 Complex via Its μ Subunit

Human immunodeficiency virus type 1 Nef provides immune evasion by decreasing the expression of major histocompatibility complex class I (MHC-I) at the surfaces of infected cells. The endosomal clathrin adaptor protein complex AP-1 is a key cellular cofactor for this activity, and it is recruited to the MHC-I cytoplasmic domain (CD) in the presence of Nef by an uncharacterized mechanism. To determine the molecular basis of this recruitment, we used an MHC-I CD-Nef fusion protein to represent the MHC-I CD/Nef complex during protein interaction assays. The MHC-I CD had no intrinsic ability to bind AP-1, but it conferred binding activity when fused to Nef. This activity was independent of the canonical leucine-based AP-binding motif in Nef; it required residue Y320 in the MHC-I CD and residues E62-65 and P78 in Nef, and it involved the mu but not the gamma/sigma subunits of AP-1. The impaired binding of mutants encoding substitutions of E62-65 or P78 in Nef was rescued by replacing the Y320SQA sequence in the MHC-I CD with YSQL, suggesting that Nef allows the YSQA sequence to act as if it were a canonical mu-binding motif. These data identify the mu subunit of AP-1 (mu1) as the key target of the MHC-I CD/Nef complex, and they indicate that both Y320 in the MHC-I CD and E62-65 in Nef interact directly with mu1. The data support a cooperative binding model in which Nef functions as a clathrin-associated sorting protein that allows recognition of an incomplete, tyrosine-based mu-binding signal in the MHC-I CD by AP-1.

Electrochemical oxidation of morin and interaction with DNA

A poly (tetrafluroethylene)-deoxyribonucleate acid (PTFE-DNA) film-modified glassy carbon electrode (GCE) has been fabricated. The electrochemical oxidation behaviors of morin as well as its interaction with DNA have been studied at PTFE-DNA film-modified GCE and bare GCE by electrochemical methods. This modified electrode shows an enhanced effectiveness towards the oxidation of morin. Importantly, as to the interaction between morin and DNA in solution, characteristic parameters such as the binding stoichiometry and association equilibrium constant according to the Hill model for cooperative binding have been determined on the basis of linear sweep voltammetry and chronocoulometry.

Crystal structure of the DNA‐binding domain of BldD, a central regulator of aerial mycelium formation in Streptomyces coelicolor A3(2)

BldD is a central regulator of the developmental process in Streptomyces coelicolor. The 1.8 angstroms resolution structure of the DNA-binding domain of BldD (BldDN) reveals that BldDN forms a compact globular domain composed of four helices (alpha1-alpha4) containing a helix-turn-helix motif (alpha2-alpha3) resembling that of the DNA-binding domain of lambda repressor. The BldDN/DNA complex model led us to design a series of mutants, which revealed the important role of alpha3 and the 'turn' region between alpha2 and alpha3 for DNA recognition. Based on the fact that BldD occupies two operator sites of bldN and whiG and shows significant disparity in the affinity toward the two operator sites when they are disconnected, we propose a model of cooperative binding, which means that the binding of one BldD dimer to the high affinity site facilitates that of the second BldD dimer to the low affinity site. In addition, structural and mutational investigation reveals that the Tyr62Cys mutation, found in the first-identified bldD mutant, can destabilize BldD structure by disrupting the hydrophobic core.

Combinatorial Identification of a Novel Consensus Sequence for the Covalent DNA-Binding Polyamide Tallimustine

Many agents successfully used in cancer chemotherapy either directly or indirectly covalently modify DNA. Examples include cisplatin, which forms a covalent adduct with guanines, and doxorubicin, which traps a cleavage intermediate between topoisomerase II and torsionally strained DNA. In most cases, the efficacy of these drugs depends on the efficiency and specificity of their DNA binding, as well as the discrimination between normal and neoplastic cells in their handling of the drug-DNA adducts. While much is known about the chemistry of drug-DNA adducts, little is known regarding the overall specificity of their formation, especially in the context of a whole human genome, where potentially billions of binding sites are possible. We used the combinatorial selection method restriction endonuclease protection, selection, and amplification (REPSA) to determine the DNA-binding specificity of the semisynthetic covalent DNA-binding polyamide tallimustine, which contains a benzoic acid nitrogen mustard appended to the minor groove DNA-binding natural product distamycin A. After investigating over 134 million possible sequences, we found that the highest affinity tallimustine binding sites contained one of two consensus sequences, either the expected distamycin hexamer binding sites followed by a CG base pair (e.g., 5'-TTTTTTC-3' and 5'-AAATTTC-3') or the unexpected sequence 5'-TAGAAC-3'. Curiously, we found that tallimustine preferentially alkylated the N7 position of guanines located on the periphery of these consensus sequences. These findings suggested a cooperative binding model for tallimustine in which one molecule noncovalently resides in the DNA minor groove and locally perturbs the DNA structure, thereby facilitating alkylation by a second tallimustine of an exposed guanine on another side of the DNA.

A new method for studying platelets, based upon the low-angle light scattering technique. 2. Application of the method in experimental toxicology and clinical pathology

A new method for studying platelets based on low-angle light scattering has been applied to studies in experimental toxicology and clinical pathology, using animal and human platelets. Index EC50for ADP within control groups was estimated to be in the range of 100–180 nmol l–1(healthy men, rabbits, rats). The refractory state of platelets was obtained by use ADP or ATP concentrations that just caused platelet activation. The time constant and the half-cycle of the refractory state of platelets were calculated. Kinetic parameters of platelet aggregation at interference of hemostasis (rabbit) and the influence of the age factors on the aggregation indices of platelets (rat) were studied. The method of low-angle light scattering was used as a diagnostic tool in experimental low-level intoxication by organophosphates and for interpretation of the pathogenesis of delayed effects. In human patients with ischemic heart disease or with prosthetic heart valves, significant changes in the functional state of platelets were observed. A model for cooperative binding of receptors with ligands on platelets from pregnant women with preeclampsia was developed.

Poly(ADP-ribose) Polymerase-1 Dimerizes at a 5‘ Recessed DNA End in Vitro: A Fluorescence Study

Activation of poly(ADP-ribose) polymerase-1 (PARP-1) is an immediate cellular reaction to DNA strand breakage as induced by alkylating agents, ionizing radiation, or oxidants. The resulting formation of protein-bound poly(ADP-ribose) facilitates survival of proliferating cells under conditions of DNA damage probably via its contribution to DNA base excision repair. In this study, we investigated the association of the amino-terminal DNA binding domain of human PARP-1 (hPARP-1 DBD) with a 5' recessed oligonucleotide mimicking a telomeric DNA end. We used the fluorescence of the Trp residues naturally occurring in the zinc finger domain of hPARP-1 DBD. Fluorescence intensity and fluorescence anisotropy measurements consistently show that the binding stoichiometry is two proteins per DNA molecule. hPARP-1 was found to bind the 5' recessed DNA end with a binding constant of approximately 10(14) M(-2) if a cooperative binding model is assumed. These results indicate that hPARP-1 DBD dimerizes during binding to the DNA target site. A footprint experiment shows that hPARP-1 DBD is asymmetrically positioned at the junction between the double-stranded and the single-stranded telomeric repeat. The largest contribution to the stability of the complex is given by nonionic interactions. Moreover, time-resolved fluorescence measurements are in line with the involvement of one Trp residue in the stacking interaction with DNA bases. Taken together, our data open new perspectives for interpretation of the selective binding of hPARP-1 to the junction between double- and single-stranded DNA.

Cooperative Regulation of Myosin-Actin Interactions by a Continuous Flexible Chain I: Actin-Tropomyosin Systems

We present a model for cooperative myosin binding to the regulated actin filament, where tropomyosins are treated as a weakly-confined continuous flexible chain covering myosin binding sites. Thermal fluctuations in chain orientation are initially required for myosin binding, leaving kinked regions under which subsequent myosins may bind without further distortion of the chain. Statistical mechanics predicts the fraction of sites with bound myosin-S1 as a function of their affinities. Published S1 binding curves to regulated filaments with different tropomyosin isoforms are fitted by varying the binding constant, chain persistence length nu (in actin monomers), and chain kink energy A from a single bound S1. With skeletal tropomyosin, we find an S1 actin-binding constant of 2.2 x 10(7) M(-1), A = 1.6 k(B)T and nu = 2.7. Similar persistence lengths are found with yeast tropomyosin. Larger values are found for tropomyosin-troponin in the presence of calcium (nu = 3.7) and tropomyosins from smooth muscle and fibroblasts (nu = 4.5). The relationship of these results to structural information and the rigid-unit model of McKillop and Geeves is discussed.

Measurement of rate constants for reactions of O2, CO, and NO with hemoglobin.

The kinetics of O2, CO, and NO binding to mammalian hemoglobins (Hbs) have been studied for 75 yr, starting with the original rapid mixing experiments of Hartridge and Roughton (1). Over the last 20 yr, these measurements have been extended to time scales ranging from hours to picoseconds. Numerous articles have been written about rapid mixing and photolysis instruments, methods for defining specific association and dissociation rate constants, and algorithms for analyzing the results in terms of specific models for cooperative ligand binding (2–10). A comprehensive review of these techniques and methods, however, is beyond the scope of this book. Instead, a practical guide to determining rate constants for O2, CO, and NO binding to native and recombinant Hbs is presented, with a special emphasis on tetrameric adult human Hb (HbA).

Structural and thermodynamic features of complexes formed by DNA and synthetic polynucleotides with dodecylamine and dodecyltrimethylammonium bromide

Complex formation of native and denatured DNA, single-stranded polyribonucleotides poly(A) and poly(U), as well as double-stranded poly(A)·poly(U) with dodecylamine (DDA) and dodecyltrimethylammonium bromide (DTAB) has been studied by UV-, CD-, IR-spectroscopy and fluorescence analysis of hydrophobic probe pyrene. DDA and DTAB were shown to bind cooperatively with DNA and polyribonucleotides, resulting in the formation of complexes containing hydrophobic micelle-like clusters. Critical aggregation concentration (CAC) of DDA and DTAB shifts sharply to lower values (30–50 times) in the presence of DNA and polynucleotides as compared to critical micelle concentration (CMC) of free DDA and DTAB in solution. The analysis of binding isotherms within the frame of the model of cooperative binding of low-molecular ligands to linear polymers allowed us to determine the thermodynamic parameters of complex formation and estimate the contribution of electrostatic interaction of positively charged heads of amphiphiles with negatively charged phosphate groups of DNA and polyribonucleotides, and hydrophobic interaction of aliphatic chains to complex stability. Electrostatic interaction was shown to make the main contribution to the stability of DNA complexes with DDA, while preferential contribution of hydrophobic interactions is characteristic of DTAB complexes with DNA. The opposite effect of DDA and DTAB on the thermal stability of DNA double helix was demonstrated from UV-melting of DNA—while DTAB stabilizes the DNA helix, DDA, to the contrary, destabilizes it. The destabilizing effect of DDA seems to originate from the displacement of intramolecular hydrogen bonds in complementary Watson–Crick A·T and G·C base pairs with intermolecular H-bonds between unsubstituted DDA amino groups and proton-accepting sites of nucleic bases.

Adsorption of Fluorescent Dyes on Oxide Nanoparticles Studied by Fluorescence Correlation Spectroscopy

In the present work, we have studied the adsorption of Rhodamine 6G on negative colloidal particles (Silica-Bindzil, Ludox-SM30 and Ludox-HS30) and Calcein on positive colloidal particles (Ludox-CL and Alumina) at very low concentrations by use of fluorescent correlation spectroscopy (FCS). In this relation we have introduced a set of equations which take into account the different quantum yield of the dye molecules and the particles as well as the adsorption of the dye molecules on the sample cell. The concentrations of fluorescent dye were varied between 2 × 10-9 and 2 × 10-7 M, and the concentrations of particles were in the range of 0.01−0.03 g/L. The adsorption data fit well with a Langmuir adsorption isotherm. The values of the adsorption constants at pH 5.5−5.7, ionic strength NaCl 1 mM, and room temperature (22 ± 0.5 °C) were in the range (0.2−2.5) × 108 L/mol, and the adsorption energy varied between −10 and 13 kJ/mol. The cooperative binding model (generalized Freundlich) was also checked but showed less satisfactory results. This work demonstrates the high performance of the FCS method for investigation of the adsorption equilibrium at solid−liquid interface. In particular the technique does not required any separation step of the adsorbed and nonadsorbed species and is not influenced by the adsorption on the sample cell walls which is often a source of artifacts when working with very low concentrations.

Thermodynamic Effects of Alcohol Additives on the Cooperative Binding of Sodium Dodecyl Sulfate to a Cationic Polymer

The binding isotherms for the interaction of sodium dodecyl sulfate (SDS) with poly(diallyldimethylammonium chloride) in aqueous solutions of 1,8-octanediol, 1,2-octanediol, or 1-butanol (BuOH) were determined by potentiometry, using a functional membrane responsive to anionic surfactants. Each alcohol caused the binding isotherm to shift to a lower SDS concentration. The magnitude of the effect was 1,2-octanediol > 1,8-octanediol ∼ BuOH. The binding isotherms were analyzed by a linear lattice model for cooperative binding. The effect of the alcohols was analyzed by thermodynamic treatment of mixtures based on the regular mixing model, and the interchange energies between alcohol and water (4.06 kT for 1,8-octanediol, 4.82 kT for 1,2-octanediol, and 3.63 kT for BuOH) and between alcohol and SDS (-1.21 kT for 1,8-octanediol, -3.52 kT for 1,2-octanediol, and -1.65 kT for BuOH) were estimated. The pronounced effect of 1,2-octanediol on the binding isotherms was ascribed to its instability in the water phase and its strong hydrophobic interaction with the SDS anion in the polyion domain. The effect of 1,8-octanediol was comparable to that of BuOH, but 1,8-octanediol was more unstable in both the water phase and the polyion domain by about kT In 2 compared to BuOH. This phenomenon can be attributed to the fact that binding of one 1,8-octanediol molecule structurally approximates the binding of two BuOH molecules.

Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest.

The coordinated termination of DNA replication is an important step in the life cycle of bacteria with circular chromosomes, but has only been defined at a molecular level in two systems to date. Here we report the structure of an engineered replication terminator protein (RTP) of Bacillus subtilis in complex with a 21 base pair DNA by X-ray crystallography at 2.5 A resolution. We also use NMR spectroscopic titration techniques. This work reveals a novel DNA interaction involving a dimeric 'winged helix' domain protein that differs from predictions. While the two recognition helices of RTP are in close contact with the B-form DNA major grooves, the 'wings' and N-termini of RTP do not form intimate contacts with the DNA. This structure provides insight into the molecular basis of polar replication fork arrest based on a model of cooperative binding and differential binding affinities of RTP to the two adjacent binding sites in the complete terminator.

PH-dependent Self-association of Influenza Hemagglutinin Fusion Peptides in Lipid Bilayers

We have recently designed a host-guest peptide system that allows us to quantitatively measure the energetics of interaction of viral fusion peptides with lipid bilayers. Here, we show that fusion peptides of influenza hemagglutinin reversibly associate with one another at membrane surfaces above critical surface concentrations, which range from one to five peptides per 1000 lipids in the systems that we investigated. It is further demonstrated by using circular dichroism and Fourier transform infrared spectroscopy that monomeric peptides insert into the bilayers in a predominantly α-helical conformation, whereas self-associated fusion peptides adopt predominantly antiparallel β-sheet structures at the membrane surface. The two forms are readily interconvertible and the equilibrium between them is determined by the pH and ionic strength of the surrounding solution. Lowering the pH favors the monomeric α-helical conformation, whereas increasing the ionic strength shifts the equilibrium towards the membrane-associated β-aggregates. The binding data are interpreted in terms of a cooperative binding model that yields free energies of insertion and free energies of self-association for each of the peptides studied at pH 7.4 and pH 5. At pH 5 and 35mM ionic strength, the insertion energy of the 20 residue influenza hemagglutinin fusion peptide is −7.2kcal/mol and the self-association energy is −1.9kcal/mol. We propose that self-association of fusion peptides could be a major driving force for recruiting a small number of hemagglutinin trimers into a fusion site.

Binding of Photosurfactant (4-butyl-azobenzene-4′-oxyethyltrimethylammonium bromide) to Polyelectrolyte

The binding of a photosurfactant, 4-butyl-azobenzene-4′-oxyethyltrimethyl ammonium bromide (BZTABr), to a linear polymer, sodium poly(2-acrylamide-2-methylpropane sulfonate) (PAMPS), has been studied by using a surfactant selective electrode. The photosurfactant undergoes atrans–cistransition on UV/vis irradiation. The binding behavior changes by varying the composition of thetransandcisforms in the solution. The ideal mixing–ideal cooperative binding model we had proposed previously for the binding of binary surfactants was applied to successfully predict the binding isotherms and the critical aggregation concentrations of the present systems. The binding of atrans–cismixture of BZTABr to a polymer is analogous to the ideal mixed micelle formation. A sort of synergetic effect was found in the binding process.

The interaction of mixed surfactants with polyelectrolytes

The interactions between a linear polymer, sodium poly(2-acrylamide-2-methylpropane sulfonate), and two cationic surfactants, dodecylpyridinium chloride and tetradecylpyridinium chloride and their mixtures with different ratios, were studied by a potentiometric titration method using a surfactant-selective electrode. The ideal mixing/ideal cooperative binding model we had proposed previously was applied to successfully predict the binding isotherms of the mixed surfactant systems and the critical aggregation concentrations of the binding. The binding of surfactant mixtures to polymers is similar to the ideal mixed micelle formation and a sort of synergetic effect was found during the binding process.

The cytosolic class II chaperonin CCT recognizes delineated hydrophobic sequences in its target proteins.

The nonhomologous proteins actin and alpha- and beta-tubulin need the assistance of the cytosolic chaperonin containing TCP-1 (CCT) to reach their correct native state, and their folding requires a transient binary complex formation with CCT. We show that separate or combined deletion of three delineated hydrophobic sequences in actin disturbs the interaction with CCT. These sites are situated between residues 125-179, 244-285, and 340-375. Also, alpha- and beta-tubulin contain at least one recognition region, and intriguingly, it has a similar distribution of hydrophobic residues as region 244-285 in actin. Internal deletion of the sites in actin favor a model for cooperative binding of target proteins to CCT. Peptide mimetics, representing the binding regions, inhibit target polypeptide binding to CCT, suggesting that actin and tubulin contact similar CCT subunits. In addition, we show that actin recognition by class II chaperonins is different from that by class I.

An “In Vitro” System Simulates in Membranes the Antibacterial Mechanism Postulated for the Action of Isoxazolylnaphtoquinoneimine inStaphylococcus aureus

The 2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naph- thoquinone-4-imine (Q1) revealed good activity againstStaphylococcus aureus.Q1in contact with the bacteria experimented reduction evidenced by changes in its spectrum of absorption simultaneously with loss of colour. During the first 4 hours of incubation, oxygenation restored the original spectrum. Treatment with sodium borohydrure reduces irreversibly Q1. Redox-reaction “in vitro” was detected between Q1and NADH in the presence of diaphorase. The environment of the probable site of action of Q1was simulated using an artificial membrane system, instead ofS. aureusmembranes. Q1interacts with lisophosphatidylcholine micelles following a cooperative binding model. The kinetics of Q1-reduction was increased by lipid micelles incorporated with the antibacterial compound.