Binding of n-mers to one-dimensional lattices with longer than close-contact interactions

A general formalism is derived for the evaluation of binding isotherms of n-mers (ligands) to one-dimensional polymers in the presence of ligand-ligand interactions which extend over several binding sites with distance-dependent interaction energies (multi-parameter model). This is an extension of the usual n-mer binding theory developed by several investigators in which ligand-ligand interaction occurs only when two ligands are in close contact (one-parameter model). The difference in binding isotherms between a one-parameter model and a multi-parameter model is studied numerically using the present formalism.     

How to develop globular proteins into adhesives

To make globular proteins suitable for application in adhesives, the specific bonds and interactions which shape their structure have to broken. Only then, a layer of relatively large, flexible and interwoven polymer chains, which are firmly attached to the solid surface by adsorption, can be created. Such a network layer is essential to save the adhesive bond under an applied force, because it can distribute the concentration of stresses generated at the interface into the bulk. Unfolding and swelling of a protein can be achieved by changing the solvent quality. For the globular whey protein beta-lactoglobulin, the optimal conditions for unfolding and swelling is found with 98% formic acid as a solvent. In formic acid, beta-lactoglobulin looses its amphoteric character (it is protonated, probably for approximately 20%). In addition, formic acid is less polar than water and thus a better solvent for the apolar parts of the protein. The swelling and unfolding behaviour of beta-lactoglobulin is studied by viscosity and CD-spectroscopy measurements. For the interpretation of the results we apply the Kuhn formalism that the conformation of a protein can be described in terms of a statistical chain which consists of segments of an average persistence length P. The statistical segment length P, which varies with the experimental conditions, is directly related to the adsorption energy required for a strong adhesion between coil and surface. It determines the depletion energy kT P(-2) m(-2) which must be overcome by specific attraction between side groups of the protein chain and the surface. For beta-lactoglobulin in 98% formic acid, we find a P value of approximately 2.2 nm, pointing at a relatively flexible chain. The minimum net adsorption energy kT P(-2) is then approximately 1 mJ m(-2), a relatively small value to be exceeded. Preliminary results of destructive adhesion tests on beech wood lap-shear joints reveal promising tensile strengths of approximately 2.9+/-1.1 N mm(-2), indeed.     

Use of binding site neighbor-effect parameters to evaluate the interactions between adjacent ligands on a linear lattice. Effects on ligand-lattice association.

A method using binding site "neighbor-effect" parameters (NEPs) is introduced to evaluate the effects of interaction between adjacent ligands on their binding to an infinite linear lattice. Binding site overlap is also taken into account. This enables the conditional probability approach of McGhee & von Hippel to be extended to more complex situations. The general equation for the isotherm is v/LF = SFKF, where v is the ratio of bound ligands to lattice residues, LF is the free ligand concentration, SF is the fraction of binding sites that are free, and KF is the average association constant of a free site. Solutions are derived for three cases: symmetric ligands, and asymmetric ligands on isotropic or anisotropic lattices. For symmetric ligands there is one NEP, E, which is the ratio of the average binding affinity of a free site if the status of the lattice residue neighboring one end of the site is unspecified (left to chance) to the affinity when this residue is free (holding the other neighbor constant). Thus KF is KE2, where K is the affinity of an isolated site. If a site is n residues long, SF is f ffn-1, where f = 1 - nv is the fraction of residues that are free and ff is the conditional probability that a free residue is bordered on a given side by another free residue. The expression for ff is 1/(1 + x/E), where x is v/f, E is (1 - x + [(1 - x)2 + 4x omega]1/2)/2, and omega is the co-operativity parameter. The binding of asymmetric ligands to an isotropic lattice is described by two NEPs; the last case involves four NEPs and a bound ligand orientation parameter. For each case, the expected length distribution of clusters of bound ligands can be calculated as a function of v. When Scatchard plots with the same intercepts and initial slope are compared, it is found that ligand asymmetry lowers the isotherm (relative to the corresponding symmetric ligand isotherm), whereas lattice anisotrophy raises it.     

Energy of adhesion of human T cells to adsorption layers of monoclonal antibodies measured by a film trapping technique.

A novel method for studying the interaction of biological cells with interfaces (e.g., adsorption monolayers of antibodies) is developed. The method is called the film trapping technique because the cell is trapped within an aqueous film of equilibrium thickness smaller than the cell diameter. A liquid film of uneven thickness is formed around the trapped cell. When observed in reflected monochromatic light, this film exhibits an interference pattern of concentric bright and dark fringes. From the radii of the fringes one can restore the shape of interfaces and the cell. Furthermore, one can calculate the adhesive energy between the cell membrane and the aqueous film surface (which is covered by a layer of adsorbed proteins and/or specific ligands), as well as the disjoining pressure, representing the force of interaction per unit area of the latter film. The method is applied to two human T cell lines: Jurkat and its T cell receptor negative (TCR-) derivative. The interaction of these cells with monolayers of three different monoclonal antibodies adsorbed at a water-air interface is studied. The results show that the adhesive energy is considerable (above 0.5 mJ/m2) when the adsorption monolayer contains antibodies acting as specific ligands for the receptors expressed on the cell surface. In contrast, the adhesive energy is close to zero in the absence of such a specific ligand-receptor interaction. In principle, the method can be applied to the study of the interaction of a variety of biological cells (B cells, natural killer cells, red blood cells, etc.) with adsorption monolayers of various biologically active molecules. In particular, film trapping provides a tool for the gentle micromanipulation of cells and for monitoring of processes (say the activation of a T lymphocyte) occurring at the single-cell level.     

Adsorption of phytochrome on several substituted sepharose gels

Phytochrome from Avena sativa shows strong adsorption with hydrophobic ligands such as octyl and phenyl Sepharose. The same behaviour was observed for undegraded (MW 400 000) and degraded (MW 60 000) phytochromes in the P_r, or P_fr, form as well. The pigment is photoreversible after adsorption on those gels. Chromatography with amino acid ligands of degraded phytochrome was also tested. The chromoprotein showed the same strong adsorption on tryptophyl Sepharose. A more specific adsorption could be achieved on histidyl Sepharose but with loss of 70% of photoreversibility. This can be interpreted by the accessibility and perturbation of the chromophoric site by the histidyl ligands     

Surface charging by large multivalent molecules. Extending the standard Gouy-Chapman treatment.

Traditionally, Gouy-Chapman theory has been used to calculate the distribution of ions in the diffuse layer next to a charged surface. In recent years, the same theory has found application to adsorption (incorporation, partitioning) of charged peptides, hormones, or drugs at the membrane-water interface. Empirically it has been found that an effective charge, smaller than the physical charge, must often be used in the Gouy-Chapman formula. In addition, the large size of these molecules can be expected to influence their adsorption isotherms. To improve evaluation techniques for such experiments, comparatively simple extensions of the standard Gouy-Chapman formalism have been studied which are based on a discrete charge virial expansion. The model allows for the mobility of charged groups at the interface. It accounts for finite size of the adsorbed macromolecules and for discrete charge effects arising from pair interactions in the interface plane. In contrast to previous discrete charge treatments this model nearly coincides with the Gouy-Chapman formalism in the case where the adsorbing molecules are univalent. Large discrepancies are found for multivalent molecules. This could explain the reduced effective charges needed in the standard Gouy-Chapman treatment. The reduction factor can be predicted. The model is mainly limited to low surface coverage, typical for the adsorption studies in question.     

Kinetics of adsorption of extended ligands on DNA at small fillings

In the present work, the adsorption kinetics of extended ligands on DNA duplexes at small fillings when molecules of DNA duplexes are on the underlayer within diffusion layer has been investigated. Both diffusion of ligands in solution (diffusion stage) and adsorption of ligands (kinetic stage) are taken into consideration at adsorption of ligands on DNA duplexes. Nonlinear system of differential equations describing adsorption of ligands where not only diffusion stage but also kinetic stage is taken into account, is obtained, moreover the equations allow localizing duplexes in arbitrary place within diffusion layer. Numeric solution of the equations makes possible to investigate the filling kinetics of DNA duplexes by ligands depending on parameters controlling adsorption process. It has been shown that depending on relation between adsorption parameters different kinetic regimes of adsorption – kinetic, complex, and diffusion regimes may be realized.     

Isotherm of ligand adsorption on DNA at multiplicative noise

Fluctuations of the number of ligands adsorbed on macromolecules are considered in the case when the number of ligands in the solution fluctuates under the action of fluctuations of the external medium (external noise). For the case of small filling, the multiplicative type of stochastic differential equation is obtained, describing the time variation of the number of ligands adsorbed on macromolecules. The isotherm of adsorption of ligands on DNA is obtained. It is shown that at small ligand concentrations, for some relations between adsorption parameters and the intensity of the external noise, no macromolecule adsorption of ligands takes place.     

Adsorption of ligands on macromolecules in the fluctuating medium

In the present work fluctuations of number of ligands adsorbed on macromolecule are investigated. We have taken into account the adsorption and desorption of ligands under the circumstance of some adsorption centers fluctuations affected by medium fluctuation. The correlation function and spectral density of number of ligands adsorbed on macromolecule are calculated. The properties of these fluctuations which allow identifying a noisemaker are determined. It has been shown, thatfas andsluggis adsorption can be distinguished by properties of dispersion and spectral density. It has been also shown, that comparison of experimental and theoretical correlation functions (or spectral densities) allows to calculate constants of ligand - adsorption center binding and unbinding.     

Equilibrium binding of derivatives of the carcinogen, benzo(a)pyrene, to DNA. Thermodynamic analysis

The physical binding of polycyclic aromatic hydrocarbon derivatives which are ultimate carcinogens to DNA may play a role in the formation of covalent DNA adducts by these compounds or in the detoxification of the compounds via DNA-catalyzed hydrolysis. Previous studies of DNA-binding interactions of derivatives of benzo(a)pyrene (BP) have been confined to low r values (r - ligands bound/base pair). We have now applied the Scatchard formalism (as modified to include neighbor exclusion) to the spectrophotometric determination of the binding of two derivatives of BP, trans - 9,10 - dihydroxydihydro - BP and 7r,8t - dihydroxy-9t,10t-oxy-7,8, 9,10-tetrahydro-BP, to double-stranded DNA at reasonably high r values. Exclusion parameters, binding constants, and thermodynamic parameters are all within the ranges found for other intercalants. Although these ligands are uncharged, the binding exhibits significant ionic strength dependence which can be rationalized (partially) by polyelectrolyte theory. Using the measured ionic strength dependence, a thermodynamic association constant, independent of ionic interactions, can be calculated which is very close to the calculated thermodynamic association constants for ethidium and proflavine.     

Is allostery an intrinsic property of all dynamic proteins?

Allostery involves coupling of conformational changes between two widely separated binding sites. The common view holds that allosteric proteins are symmetric oligomers, with each subunit existing in "at least" two conformational states with a different affinity for ligands. Recent observations such as the allosteric behavior of myoglobin, a classical example of a nonallosteric protein, call into question the existing allosteric dogma. Here we argue that all (nonfibrous) proteins are potentially allosteric. Allostery is a consequence of re-distributions of protein conformational ensembles. In a nonallosteric protein, the binding site shape may not show a concerted second-site change and enzyme kinetics may not reflect an allosteric transition. Nevertheless, appropriate ligands, point mutations, or external conditions may facilitate a population shift, leading a presumably nonallosteric protein to behave allosterically. In principle, practically any potential drug binding to the protein surface can alter the conformational redistribution. The question is its effectiveness in the redistribution of the ensemble, affecting the protein binding sites and its function. Here, we review experimental observations validating this view of protein allostery.     

Short-range interactions and size of ligands bound to DNA strongly influence adsorptive phase transition caused by long-range interactions

Long-range interaction between all the ligands bound to DNA molecule may give rise to adsorption with the character of phase transition of the first kind (D. Y. Lando, V. B. Teif, J. Biomol. Struct & Dynam. 18, 903-911 (2000)). In this case, the binding curve, c(c(o)), is characterized by a sudden change of the relative concentration of bound ligands ((c)) at a critical concentration of free (unbound) ligands, c(o)=c(ocr), from a low c value to a high one where c(o) is molar concentration of free ligands. Such a transition might be caused by some types of DNA condensation or changes in DNA topology. For the study of the conditions necessary for adsorption with the character of phase transition, a calculation procedure based on the method of the free energy minimum is developed. The ligand size and two types of interactions between ligands adsorbed on DNA molecule are taken into consideration: long-range interaction between all the ligands bound to DNA and contact interactions between neighboring ligands. It was found that a) Stronger long-range interaction is required for longer ligands to induce phase transition that is occurred at greater c(ocr) values; b) Pure contact interaction between neighboring ligands can not itself initiate phase transition. However contact cooperativity strongly decreases the threshold value of energy of long-range interaction necessary to give rise to the transition.     

Guest Editorial

Abstract

This special issue is a selection of papers from the sessions on Molecular Simulation that were presented at the annual American Institute of Chemical Engineering meeting in Miami Beach, Florida, in November 1992. It reflects the great diversity of research that is now underway using atomic-level computer simulation methods and the progression to simulations of more complex systems. In this issue, for instance, the materials studied range from electrolytes (Chialvo and Cummings) to polymers (Broadbelt, LaMarca, Klein, Andrews and Colter) to precursors to exotic natural gas hydrates (Long and Sloan). The study of solid/fluid interfaces is an emerging theme. Wang and Fichthorn have studied the adsorption of EAM modeled Pt on Pt substrates. Ravi and Murad have produced an interesting new method of studying the adsorption of fluids, here nitrogen in pores. Uttorrnark et al. have studied the kinetics of growth and dissolution processes in solidAiquid systems.     

Surface plasmon resonance as a probe of protein isomerization

This article presents new concepts in affinity chromatography/mass spectrometry for the study of molecular interactions. Chromatographic assays involving estrogen receptor-beta, sorbitol dehydrogenase, human alpha-thrombin, cholera toxin B subunit, beta-galactosidase, and Griffonia simplicifolia isolectin B(4) were established in microaffinity columns and operated in frontal analysis mode. Methods and formalism are presented for the measurement of dissociation constants, using direct methods in which the mass spectrometric signature of the ligand is used to measure breakthrough time and, hence, binding strength. The direct approach is capable of measuring sub-micromolar K(d) and higher, on sub-pmol amounts of immobilized protein, as shown in the cholera toxin assay. Indirect assays that demonstrate the advantage of routine, rugged performance were developed. By tracking the effect of a test ligand on a selected probe, or indicator ligand, dissociation constants in the low nanomolar range could be reliably determined for ligands to estrogen receptor-beta. Mass spectrometry supports the resolution of complex ligand mixtures, and it is demonstrated in the sorbitol dehydrogenase assay that ligands can be rank ordered across approximately three orders of magnitude in K(d), in a single run. A new concept for rapid mixture prescreening is presented, in which an indicator ligand can be used to discriminate between mixtures that contain high levels of weak ligands and those that contain single strong ligands.     

92 Novel interpretation of the isotherms of multimodal ligands binding with DNA

The binding of ligands with DNA is a key moment in a whole range of cellular processes that provide not only the normal cell vital activity but also the development of some pathological processes. Depending on ligand type, structure of DNA adsorption centers, and physical–chemical conditions of the surrounding, the ligand may bind to DNA by several modes [1]. Particularly, adsorption isotherm of multimodal ligands binding to DNA in Scatchard’s coordinates has a concave shape with two brightly expressed linear areas in the region of small fillings. The analysis of such type of adsorption isotherm for determining of important binding parameters such as binding constant and number of adsorption centers (the part of DNA polymer with which one ligand molecule binds) presents difficulties. Practically in all cases, the analysis of such adsorption isotherm is carried out by linear parts of curves. Such analysis mode of experimental points is approximate method, since all registered of experimental points are roughly divided into two groups and they are treated by linear binding isotherm and therefore the binding parameters are determined. In the present work, the non-linear adsorption isotherm in Scatchard‘s coordinates is obtained which allowed, provided, the more precise treatment of all experimental points by unique curve which includes linear regions as well. Such mode of treatment of experimental points makes more precise the determination of not only binding constant and number of adsorption centers that correspond to the one ligand molecule binding, but also additional binding parameter – a proportion of adsorption centers of each binding to DNA type of multimodal ligand.     

Conformational flexibility of the Cys 697-Cys 707 segment of myosin subfragment-1. Distance distributions by frequency-domain fluorometry

The separation between Cys 697 (SH1) and Cys 707 (SH2) of the heavy chain of myosin subfragment-1 was previously measured by fluorescence resonance energy transfer with a donor linked to SH1 and an acceptor to SH2. In the present study the distribution of the distances between the two thiols was recovered from frequency-domain fluorometry. In the native state and in the presence of ligands such as MgADP, pyrophosphate, orthovanadate (Vi) and actin, we found wide distributions of the separations between SH1 and SH2 (11-16 A) comparable to that found in the random-coil state (20 A). These results suggest that the SH1-SH2 segment has a high degree of conformational flexibility even in native S1. The flexibility is not much affected by the physiological state of S1. However, the ligands MgADP, Vi and MgADP + Vi decrease significantly the mean SH1-SH2 distance from 27 to 17 A with the effect of MgADP+ Vi being the most pronounced. The anisotropy decay of donor-labeled S1 is biphasic with two rotational correlation times. The long component is decreased by these ligands from 289 to 93 ns, suggesting a more compact symmetric structure of S1 in the presence of the ligands. The complex S1(MgADP)Vi has been shown to be a stable analogue of S1(MgADP)Pi, an unstable intermediate that is generated in the actomyosin ATPase cycle during muscle contraction. Since the power stroke of muscle is accompanied by release of Pi from S1(MgADP)Pi, the present results are consistent with a model in which force generation can be accompanied by transition of S1 from a highly symmetric or compact structure to a more extended structure.     

Biosorption Processes for Natural and Wastewater Treatment – Part 1: Literature Review

An analysis is made of the literature on the mechanism of biosorption‐bioregeneration of organic substances mainly on activated carbon (AC). There have advanced two hypotheses on the biosorption mechanism: the Rodman exoenzymatic hypothesis and the bioregeneration approach of Andrews and Chi Tien. In addition, it was shown that the principal mechanism responsible for the removal of both biodegradable and bioresistant compounds is biological oxidation, but only in combination with adsorption on activated carbon. The authors examine the role of the filter medium in biosorption, factors affecting efficiency of biosorption and bioregeneration of AC and technological aspects of the application of biosorptive processes.