Model analysis of surfactant--polymer interaction as cooperative ligand binding to linear lattice

An improved model of the cooperative binding of monomeric ligands to a linear lattice is proposed for the analysis of surfactant association on the polymer. The interaction between bound ligands across an unoccupied site as well as the steric hindrance effect in consecutive bindings is taken into account here. Typical results of the model calculations are represented, and several least squares fittings of the binding isotherms of the ionic surfactant-polyelectrolyte systems are attempted. The characteristic binding behavior in those systems is interpretable by the feasible model of the interactions between surfactant molecules. The advantages and limitations of the analysis using this model also are discussed.     

The structure of a polyQ-anti-polyQ complex reveals binding according to a linear lattice model

Huntington and related neurological diseases result from expansion of a polyglutamine (polyQ) tract. The linear lattice model for the structure and binding properties of polyQ proposes that both expanded and normal polyQ tracts in the preaggregation state are random-coil structures but that an expanded polyQ repeat contains a larger number of epitopes recognized by antibodies or other proteins. The crystal structure of polyQ bound to MW1, an antibody against polyQ, reveals that polyQ adopts an extended, coil-like structure. Consistent with the linear lattice model, multimeric MW1 Fvs bind more tightly to longer than to shorter polyQ tracts and, compared with monomeric Fv, bind expanded polyQ repeats with higher apparent affinities. These results suggest a mechanism for the toxicity of expanded polyQ and a strategy to link anti-polyQ compounds to create high-avidity therapeutics.     

Kinetic mechanism of rat polymerase beta-dsDNA interactions. Fluorescence stopped-flow analysis of the cooperative ligand binding to a two-site one-dimensional lattice

Kinetics of cooperative binding of rat polymerase beta to a double-stranded DNA has been studied using the fluorescence stopped-flow techniques. The data have been analyzed by an approach developed to examine complete kinetics of cooperative large ligand binding to a one-dimensional lattice. The method is based on using the smallest possible system that preserves key ingredients of cooperative binding; i.e., at saturation, the lattice can accept only two ligand molecules. It allows the identification of collective amplitudes as well as amplitudes describing particular normal modes of the reaction. The mechanism of the intrinsic binding of pol beta to the dsDNA is different from the analogous mechanism for the ssDNA. The difference originates from different enzyme orientations in the corresponding complexes. Intrinsic binding to the dsDNA includes only two sequential steps: a very fast bimolecular association followed by an energetically favorable conformational transition of the complex. The transition following the bimolecular step does not facilitate the engagement of the enzyme in cooperative interactions. Its role seems to be reinforcing the affinity of the bimolecular step. Salt and magnesium cations affect both the bimolecular step and the conformational transition. As a result, the bimolecular step is less sensitive to the increased salt concentration, allowing the enzyme to preserve its initial dsDNA affinity. The changing character of cooperative interactions between bound enzyme molecules as a function of NaCl concentration and MgCl(2) concentration does not affect the binding mechanism. The engagement in cooperative interactions is approximately 3-4 orders of magnitude slower than the conformational transition of the DNA-bound polymerase. The importance of the obtained results for the pol beta activities is discussed.     

A novel computational approach "BP-STOCH" to study ligand binding to finite lattice

We report a novel computational algorithm "BP-STOCH" to be used for studying single-type ligand binding with biopolymers of finite lengths, such as DNA oligonucleotides or oligopeptides. It is based on an idea to represent any type of ligand-biopolymer complex in a form of binary number, where "0" and "1" bits stand for vacant and engaged monomers of the biopolymer, respectively. Cycling over all binary numbers from the lowest 0 up to the highest 2(N) - 1 means a sequential generating of all possible configurations of vacant/engaged monomers, which, after proper filtering, results in a full set of possible types of complexes in solution between the ligand and the N-site lattice. The principal advantage of BP-STOCH algorithm is the possibility to incorporate into this cycle any conditions on computation of the concentrations and observed experimental parameters of the complexes in solution, and programmatic access to each monomer of the biopolymer within each binding site of every binding configuration. The latter is equivalent to unlimited extension of the basic reaction scheme and allows to use BP-STOCH algorithm as an alternative to conventional computational approaches.     

Cooperativity in ligand binding: a new graphic analysis.

When analyzing binding of ligands to macromolecules, the existence of site-site interactions complicates a straightforward interpretation of the binding parameters obtained through classical analytical methods, such as the Scatchard plot. For describing site-site interactions, we propose a new parameter, the $\text{average affinity}$ of the receptor sites, $\text{K}$, calculated as $\text{(}\text{B}\text{F}\text{)/(R}{}_{\mathrm{o}}\text{?B)}$. Plotting $\text{K}$ as a function of fractional occupancy ($\text{B}\text{R}{}_{\mathrm{o}}$), reveals that: (1) at very low occupancy a limiting high $\text{K}$ is obtained ($\text{K}$_e) (“empty sites” conformation); (2) when the fraction of sites filled increases above a certain threshold, $\text{K}$ begins to fall due to increasing site-site interactions until (3) a limiting low $\text{K}$ ($\text{K}$_f) is obtained (“filled sites” conformation). This method has been successfully applied to the negative cooperativity of insulin receptors.     

The binding of an indefinitely associating ligand to acceptor: consideration of monovalent ligand species binding to a multivalent acceptor

Currently available binding theory is extended to incorporate the concept of indefinite self-association of the ligand. Binding equations are formulated in closed form for the case of the binding to a multivalent acceptor of a ligand capable of isodesmically indefinitely self-associating in a "head-to-tail" mode such that each ligand state bears one site capable of interacting with the acceptor. It is shown both mathematically and by way of numerical example that this system will give rise exclusively to binding curves convex to the r-axis in Scatchard format. Thus, the system provides another example of a binding mechanism capable of generating an apparent negatively co-operative binding response.     

Methods for the analysis of the kinetics of cooperative binding of ligands to a two-site lattice

Solutions are obtained that describe the time dependence of the reversible binding of a ligand to a two-site lattice. The binding may be cooperative. Three methods are used to obtain these solutions: the separation of on/off processes with a variable transformation, the asymptotic series analysis, and the singular perturbation procedure. Applications to parameter calculation from experimental data are presented. This kinetic system is such that it is difficult to extract all kinetic parameters from data analysis, and the implications for each method are also discussed.     

Studies of ligand binding to arrestin

A striking homology is observed between the regions 70-83 and 361-374 of the sequence of bovine arrestin and the calcium-binding loops of calmodulin and troponin C. However, the predicted alpha-helices flanking the calcium-binding site in calmodulin and troponin C are not present in arrestin. Direct measurements therefore were made in order to assess whether arrestin can bind calcium. We found that arrestin does not bind Ca2+ at physiological ionic strength, as determined by equilibrium dialysis, gel filtration, and fluorescence spectroscopy. Rapid and quantitative precipitation of arrestin occurs with Tb3+. The precipitation is reversed by EDTA and blocked by Mg2+ but not by Ca2+. Prompted by several reports, we also investigated whether nucleotides bind to arrestin. Neither ATP nor GTP binds under the conditions tested. Binding of arrestin to photolyzed, phosphorylated rhodopsin also does not influence the binding of calcium or nucleotides.     

Kinetics of ligand binding to haemoproteins

A mass of experimental data has been accumulated in the 65 years since Hartridge and Roughton made the first measurement of the rapid reaction of haemoglobin with O2 in solution on a millisecond time scale, at first by flow-mixing methods, and, for 30 years or so, by flash photolysis. Technical advances, particularly in lasers, have allowed increasingly rapid reactions to be followed and the fastest reactions now observed have half-times conveniently measured in pico-seconds. The measurements were used at first to discuss the physiology of gas transport and to describe co-operativity in haemoglobin. More recently, the process of ligand binding has been dissected into intramolecular and intermolecular components. Relating the various rates to the abundance of structural information on crystals is so difficult that the work has barely begun, but the combination of kinetic measurements with genetic engineering and crystallography has promise, as well as problems, for the future.     

Kinetics of ligand binding to a cluster of membrane-associated receptors

The process of ligand binding to a cluster of membrane-associated receptors is examined theoretically. The theoretical model proposed involves the diffusion of ligands from the solution to the disc-like cluster of receptors on the surface of the spherical cell. When the ligand hits the internal part of the disc-like cluster, it begins to move laterally until it leaves the disc through its outer surface or is bound by one of the receptors inside the disc. If the ligand leaves the cluster, it returns to the solution and hits the disc again after a certain period, etc. According to our model the transition from a diffusion-limited to a reaction-limited process of binding is determined by the dimensionless parameter Dt _c/a ², where D is the lateral diffusion coefficient,t _c is the characteristic time of reaction, anda is the radius of the disc-like cluster. The forward rate constantk _f turns out to be a function of . Comparing the results of our calculations ofk _f with some experimental data we found that agreement is achieved at high , i.e. the process of ligand binding by clustered receptors is predominantly reaction-limited.     

Self-consistent theory of reversible ligand binding to a spherical cell

Ligand binding to receptors is the initial event in many signaling processes, and a quantitative understanding of this interaction is important for modeling cell behavior. In this paper, we study the kinetics of reversible ligand binding to receptors on a spherical cell surface using a self-consistent stochastic theory. Binding, dissociation, diffusion and rebinding of ligands are incorporated into the theory in a systematic manner. We derive explicitly the time evolution of the ligand-bound receptor fraction p(t) in various regimes. Contrary to the commonly accepted view, we find that the well-known Berg-Purcell scaling for the association rate is modified as a function of time. Specifically, the effective on-rate changes non-monotonically as a function of time and equals the intrinsic rate at very early as well as late times, while being approximately equal to the Berg-Purcell value at intermediate times. The effective dissociation rate, as it appears in the binding curve or measured in a dissociation experiment, is strongly modified by rebinding events and assumes the Berg-Purcell value except at very late times, where the decay is algebraic and not exponential. In equilibrium, the ligand concentration everywhere in the solution is the same and equals its spatial mean, thus ensuring that there is no depletion in the vicinity of the cell. Implications of our results for binding experiments and numerical simulations of ligand-receptor systems are also discussed.     

Preferential binding of a G-quadruplex ligand to human chromosome ends

The G-overhangs of telomeres are thought to adopt particular conformations, such as T-loops or G-quadruplexes. It has been suggested that G-quadruplex structures could be stabilized by specific ligands in a new approach to cancer treatment consisting in inhibition of telomerase, an enzyme involved in telomere maintenance and cell immortality. Although the formation of G-quadruplexes was demonstrated in vitro many years ago, it has not been definitively demonstrated in living human cells. We therefore investigated the chromosomal binding of a tritiated G-quadruplex ligand, ³H-360A (2,6-N,N′-methyl-quinolinio-3-yl)-pyridine dicarboxamide [methyl-³H]. We verified the in vitro selectivity of ³H-360A for G-quadruplex structures by equilibrium dialysis. We then showed by binding experiments with human genomic DNA that ³H-360A has a very potent selectivity toward G-quadruplex structures of the telomeric 3′-overhang. Finally, we performed autoradiography of metaphase spreads from cells cultured with ³H-360A. We found that ³H-360A was preferentially bound to chromosome terminal regions of both human normal (peripheral blood lymphocytes) and tumor cells (T98G and CEM1301). In conclusion, our results provide evidence that a specific G-quadruplex ligand interacts with the terminal ends of human chromosomes. They support the hypothesis that G-quadruplex ligands induce and/or stabilize G-quadruplex structures at telomeres of human cells.     

NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase

Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases.     

A pre-steady state analysis of ligand binding to human glucokinase: evidence for a preexisting equilibrium

Cooperativity with glucose is a key feature of human glucokinase (GK), allowing its crucial role as a glucose sensor in hepatic and pancreatic cells. We studied the changes in enzyme intrinsic tryptophan fluorescence induced by binding of different ligands to this monomeric enzyme using stopped-flow and equilibrium binding methods. Glucose binding data under pre-steady state conditions suggest that the free enzyme in solution is in a preexisting equilibrium between at least two conformers (super-open and open) which differ in their affinity for glucose (Kd* = 0.17 +/- 0.02 mM and Kd = 73 +/- 18 mM). Increasing the glucose concentration changes the ratio of the two conformers, thus yielding an apparent Kd of 3 mM (different from a Km of 7-10 mM). The rates of conformational transitions of free and GK complexed with sugar are slow and during catalysis are most likely affected by ATP binding, phosphate transfer, and product release steps to allow the kcat to be 60 s-1. The ATP analogue PNP-AMP binds to free GK (super-open) and GK-glucose (open) complexes with comparable affinities (Kd = 0.23 +/- 0.02 and 0.19 +/- 0.08 mM, respectively). However, cooperativity with PNP-AMP observed under equilibrium binding conditions in the presence of glucose (Hill slope of 1.6) is indicative of further complex tightening to the closed conformation. Another physiological modulator (inhibitor), palmitoyl-CoA, binds to GK with similar characteristics, suggesting that conformational changes induced upon ligand binding are not restricted by an active site ligand. In conclusion, our data support control of GK activity and Km through the ratio of distinct conformers (super-open, open, and closed) through either substrate or other ligand binding and/or dissociation.     

Enthalpies of ligand binding to bovine neurophysins

Flow microcalorimetry and batch microcalorimetry have been used to survey the energetics of ligand binding by bovine neurophysins I and II. Calorimetry studies were supplemented by van't Hoff analyses of binding constants determined by circular dichroism. Free energies of binding of a series of di- and tripeptides that bind to the strong hormone binding site of neurophysin were partitioned into their enthalpic and entropic components. The results indicate that, at 25 degrees C, the binding of most peptides is an enthalpy-driven reaction associated with negative entropy and heat capacity changes. Studies elsewhere, supported by evidence here, indicate that the principal component of the negative enthalpy change does not arise from the increase in neurophysin dimerization associated with peptide binding. Accordingly, the negative enthalpy change is attributed to direct bonding interactions with peptide and possibly also to peptide-induced changes in tertiary or quaternary organization. Comparison of the binding enthalpies of different peptides indicated two types of bonding interactions that contribute to the negative enthalpy change of peptide ligation. Substitution of an aromatic- or sulfur-containing side chain for an aliphatic side chain in position 1 of bound peptides led to increases in negative enthalpy of from 1 to 6 kcal/mol, demonstrating that interactions typically classified as hydrophobic can have a significant exothermic component at 25 degrees C. Similarly, loss of hydrogen bonding potential in the peptide decreased the enthalpy change upon binding, in keeping with the expected enthalpic contribution of hydrogen bonds. In particular, the data suggested that the peptide backbone between residues 2 and 3 and the phenolic hydroxyl group in position 2 participate in hydrogen bonding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamics of ligand binding to myoglobin.

Myoglobin rebinding of carbon monoxide and dioxygen after photodissociation has been observed in the temperature range between 40 and 350 K. A system was constructed that records the change in optical absorption at 436 nm smoothly and without break between 2 musec and 1 ksec. Four different rebinding processes have been found. Between 40 and 160 K, a single process is observed. It is not exponential in time, but approximately given by N(t) = (1 + t/to)-n, where to and n are temperature-dependent, ligand-concentration independent, parameters. At about 170 K, a second and at 200 K, a third concentration-independent process emerge. At 210 K, a concentration-dependent process sets in. If myoglobin is embedded in a solid, only the first three can be seen, and they are all nonexponential. In a liquid glycerol-water solvent, rebinding is exponential. To interpret the data, a model is proposed in which the ligand molecule, on its way from the solvent to the binding site at the ferrous heme iron, encounters four barriers in succession. The barriers are tentatively identified with known features of myoglobin. By computer-solving the differential equation for the motion of a ligand molecule over four barriers, the rates for all important steps are obtained. The temperature dependences of the rates yield enthalpy, entropy, and free-energy changes at all barriers. The free-energy barriers at 310 K indicate how myoglobin achieves specificity and order. For carbon monoxide, the heights of these barriers increase toward the inside; carbon monoxide consequently is partially rejected at each of the four barriers. Dioxygen, in contrast, sees barriers of about equal height and moves smoothly toward the binding site. The entropy increases over the first two barriers, indicating a rupturing of bonds or displacement of residues, and then smoothly decreases, reaching a minimum at the binding site. The magnitude of the decrease over the innermost barrier implies participation of heme and/or protein. The nonexponential rebinding observed at low temperatures and in solid samples implies that the innermost barrier has a spectrum of activation energies. The shape of the spectrum has been determined; its existence can be explained by assuming the presence of many conformational states for myoglobin. In a liquid at temperatures above about 230 K, relaxation among conformational states occurs and rebinding becomes exponential.