Probabilistic description of the system "ligand-receptor"

The theory of probabilities was used to describe the ligand-receptor interaction. Mean and variance of number ligand-receptor complexes are calculated. It is shown that the mean number of ligand-receptor complexes coincides with that obtained from the law of conservation masses. Proceeding from a ratio of mean and expectation it is shown that the variance of the number of ligand-receptor complexes should be taken into account with concentration of ligand-receptor complexes component less than 1 fmol.     

Nanomechanical microcantilever operated in vibration modes with use of RNA aptamer as receptor molecules for label-free detection of HCV helicase

We report the nanomechanical microcantilevers operated in vibration modes (oscillation) with use of RNA aptamers as receptor molecules for label-free detection of hepatitis C virus (HCV) helicase. The nanomechanical detection principle is that the ligand-receptor binding on the microcantilever surface induces the dynamic response change of microcantilevers. We implemented the label-free detection of HCV helicase in the low concentration as much as 100 pg/ml from measuring the dynamic response change of microcantilevers. Moreover, from the recent studies showing that the ligand-receptor binding generates the surface stress on the microcantilever, we estimate the surface stress, on the oscillating microcantilevers, induced by ligand-receptor binding, i.e. binding between HCV helicase and RNA aptamer. In this article, it is suggested that the oscillating microcantilevers with use of RNA aptamers as receptor molecules may enable one to implement the sensitive label-free detection of very small amount of small-scale proteins.     

Lanthanide-based luminescent assays for ligand-receptor interactions

The evaluation of receptor ligand interactions is important in the field of drug discovery and development. Currently these interactions are typically measured with cumbersome (low throughput) radiolabels. Higher throughput screens are available such as fluorescent measurements of G-protein coupled receptor-induced Ca2+ increases or fluorescence anisotropy, yet these have limited applicability and/or low signal to noise. Hence, there is a need to develop more widely applicable and more sensitive labels that can be used to monitor ligand-receptor interactions. Lanthanides provide an attractive alternative to the traditional labels used for monitoring ligand-receptor interactions. The incorporation of lanthanide labels into traditional assays used to assess receptor-ligand interactions can make these assays more affordable, less time consuming and amenable to automation. Lanthanides can be coupled to ligands and provide strong luminescent signals that can be detected using time-resolved fluorescence (TRF) methods. This approach takes advantage of the long fluorescence lifetime of the lanthanide and can detect less than one attomole of europium in a multiwell plate sample. This short review provides a basic introduction into lanthanides and TRF and describes some of the recent assays which have utilized lanthanides as labels to assess ligand-receptor interactions.     

Interaction between electromagnetic fields and cells: microelectrophoretic effect on ligands and surface receptors

The aggregation between lectins and lymphocyte surface receptors can be affected strongly by a low-level electric field induced in the cell suspension by a time-varying magnetic field. One of the possible mechanisms is the microelectrophoretic effect due to the electric field, which influences the distance (in the mean square sense) between charged ligands and receptors when they are about to separate. On a purely theoretical basis, it is shown that, at low frequencies, an externally induced periodic electric field always decreases the mean lifetime of ligand-receptor complexes. As a consequence, the mitogenic gain obtained by lectin addition to cell suspension is decreased. These results suggest that such a mechanism, if effective, reduces the lectin mitogenic capability and offers a way of handling similar phenomena which have been described for other biological systems.     

Stepped changes of monovalent ligand-binding force during ligand-induced clustering of integrin alphaIIB beta3

Recent evidence demonstrated that conformational changes of the integrin during receptor activation affected its binding to extracellular matrix; however, experimental assessment of ligand-receptor binding following the initial molecular interaction has rarely been carried out at a single-molecule resolution. In the present study, laser tweezers were used to measure the binding force exerted by a live Chinese hamster ovary cell that expressed integrin alphaIIb beta3 (CHO alphaIIb beta3), to the bead carrier coated with the snake venom rhodostomin that served as an activated ligand for integrin alphaIIb beta3. A progressive increase of total binding force over time was noticed when the bead interacted with the CHO alphaIIb beta3 cell; such an increase was due mainly to the recruitment of more integrin molecules to the bead-cell interface. When the binding strength exerted by a single ligand-receptor pair was derived from the "polyvalent" measurements, surprisingly, a stepped decrease of the "monovalent binding force" was noted (from 4.15 to 2.54 piconewtons (pN)); such decrease appeared to occur during the ligand-induced integrin clustering process. On the other hand, the mutant rhodostomin defective in clustering integrins exhibited only one (1.81 pN) unit binding strength.     

Stem cell-derived exosomes as a therapeutic tool for cardiovascular disease

Mesenchymal stem cells(MSCs) have been used to treat patients suffering from acute myocardial infarction(AMI) and subsequent heart failure. Although it was originally assumed that MSCs differentiated into heart cells such as cardiomyocytes, recent evidence suggests that the differentiation capacity of MSCs is minimal and that injected MSCs restore cardiac function via the secretion of paracrine factors. MSCs secrete paracrine factors in not only naked forms but also membrane vesicles including exosomes containing bioactive substances such as proteins, messenger RNAs, and microR NAs. Although the details remain unclear, these bioactive molecules are selectively sorted in exosomes that are then released from donor cells in a regulated manner. Furthermore, exosomes are specifically internalized by recipient cells via ligand-receptor interactions. Thus, exosomes are promising natural vehicles that stably and specifically transport bioactive molecules to recipient cells. Indeed, stem cell-derived exosomes have been successfully used to treat cardiovascular disease(CVD), such as AMI, stroke, and pulmonary hypertension, in animal models, and their efficacy has been demonstrated. Therefore, exosome administration may be a promising strategy for the treatment of CVD. Furthermore, modifications of exosomal contents may enhance their therapeutic effects. Future clinical studies are required to confirm the efficacy of exosome treatment for CVD.     

The complexity of complexes in signal transduction

Many activities of cells are controlled by cell-surface receptors, which in response to ligands, trigger intracellular signaling reactions that elicit cellular responses. A hallmark of these signaling reactions is the reversible nucleation of multicomponent complexes, which typically begin to assemble when ligand-receptor binding allows an enzyme, often a kinase, to create docking sites for signaling molecules through chemical modifications, such as tyrosine phosphorylation. One function of such docking sites is the co-localization of enzymes with their substrates, which can enhance both enzyme activity and specificity. The directed assembly of complexes can also influence the sensitivity of cellular responses to ligand-receptor binding kinetics and determine whether a cellular response is up- or downregulated in response to a ligand stimulus. The full functional implications of ligand-stimulated complex formation are difficult to discern intuitively. Complex formation is governed by conditional interactions among multivalent signaling molecules and influenced by quantitative properties of both the components in a system and the system itself. Even a simple list of the complexes that can potentially form in response to a ligand stimulus is problematic because of the number of ways signaling molecules can be modified and combined. Here, we review the role of multicomponent complexes in signal transduction and advocate the use of mathematical models that incorporate detail at the level of molecular domains to study this important aspect of cellular signaling.     

High-sensitivity cytofluorometric quantitation of lectin and hormone binding to surfaces of living cells.

With a specially equipped flow cytofluorometer it is possible to determine quickly and accurately binding constants and the maximum number of binding sites for ligands such as peptide hormones and lectins on surfaces of intact living cells, with incubation concentrations as low as 10^?11 M. Since the measurement is confined to cell-bound material the cells can be kept in their physiological environment, including free ligand molecules, even at the very moment of the assay. Thus there is no additional risk of perturbing the integrity of the membrane or of interfering with ligand-receptor interactions by washing or similar procedures. It was found that damaged cells, inevitably present in any population, are able to grossly distort binding patterns. Suggestions are given how such cells may be excluded from the measurement.     

A Tunable Coarse-Grained Model for Ligand-Receptor Interaction

Cell-surface receptors are the most common target for therapeutic drugs. The design and optimization of next generation synthetic drugs require a detailed understanding of the interaction with their corresponding receptors. Mathematical approximations to study ligand-receptor systems based on reaction kinetics strongly simplify the spatial constraints of the interaction, while full atomistic ligand-receptor models do not allow for a statistical many-particle analysis, due to their high computational requirements. Here we present a generic coarse-grained model for ligand-receptor systems that accounts for the essential spatial characteristics of the interaction, while allowing statistical analysis. The model captures the main features of ligand-receptor kinetics, such as diffusion dependence of affinity and dissociation rates. Our model is used to characterize chimeric compounds, designed to take advantage of the receptor over-expression phenotype of certain diseases to selectively target unhealthy cells. Molecular dynamics simulations of chimeric ligands are used to study how selectivity can be optimized based on receptor abundance, ligand-receptor affinity and length of the linker between both ligand subunits. Overall, this coarse-grained model is a useful approximation in the study of systems with complex ligand-receptor interactions or spatial constraints.     

A kinetic model of the transient phase in the response of olfactory receptor neurons

A model is presented that predicts the instantaneous spike rate of an olfactory receptor neuron (ORN) in response to the quality and concentration of an odor stimulus. The model accounts for the chemical kinetics of ligand-receptor binding and activation processes, and implicitly the initiation of second messenger cascades that lead to depolarization and/or hyperpolarization of the ORN membrane. Both of these polarizing processes are included in the most general form of the model, as well as a process that restores the voltage to its negative resting state. The spike rate is assumed to be linearly proportional to the level of voltage depolarization above a critical negative voltage level. The model includes the simplifying assumption that activation of bound ligand-receptor complexes by G-proteins and other enabling molecules follows a Monod function that has the ratio of enabling molecules to bound unactivated ligand-receptor complexes as its argument. Parameters are selected that provide an excellent fit of the model to previously published empirical data on the response of cockroach ORNs to pulsed 1-hexanol stimuli. The sensitivity of model output to various model parameters is investigated and changes to parameters are discussed that would improve the ability of ORNs to follow rapidly pulsed stimuli.     

Mathematical modeling the kinetics of cell distribution in the process of ligand-receptor binding

A statistical approach is presented to model the kinetics of cell distribution in the process of ligand-receptor binding on cell surfaces. The approach takes into account the variation of the amount of receptors on cells assuming the homogeneity of monovalent binding sites and ligand molecules. The analytical expressions for the kinetics of cell distribution have been derived in the reaction-limited approximation. In order to demonstrate the applicability of the mathematical model, the kinetics of binding the rabbit, anti-mouse IgG with Ig-receptors of the murine hybridoma cells has been measured. Anti-mouse IgG was labeled with fluorescein isothiocyanate (FITC). The kinetics of cell distribution on ligand-receptor complexes was observed during the reaction process by real-time measuring of the fluorescence and light-scattering traces of individual cells with the scanning flow cytometer. The experimental data were fitted by the mathematical model in order to obtain the binding rate constant and the initial cell distribution on the amount of receptors.     

Effects of clustered epitopes in multivalent ligand-receptor interactions

Many biological ligands are composed of clustered binding epitopes. However, the effects of clustered epitopes on the affinity of ligand-receptor interactions in many cases are not well understood. Clustered carbohydrate epitopes are present in naturally occurring multivalent carbohydrates and glycoproteins, which are receptors on the surface of cells. Recent studies have provided evidence that the enhanced affinities of lectins, which are carbohydrate binding proteins, for multivalent carbohydrates and glycoproteins are due to internal diffusion of lectin molecules from epitope to epitope in these multivalent ligands before dissociation. Indeed, binding of lectins to mucins, which are large linear glycoproteins, appears to be similar to the internal diffusion mechanism(s) of protein ligands binding to DNA, which have been termed the "bind and slide" or "bind and hop" mechanisms. The observed increasing negative cooperativity and gradient of decreasing microaffinity constants of a lectin binding to multivalent carbohydrates and glycoproteins result in an initial fraction of lectin molecules that bind with very high affinity and dynamic motion. These findings have important implications for the mechanisms of binding of lectins to mucins, and for other ligand-biopolymer interactions and clustered ligand-receptor systems in general.     

Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes

The dynamics of protein distribution in endocytic membranes are relevant for many cellular processes, such as protein sorting, organelle and membrane microdomain biogenesis, protein-protein interactions, receptor function, and signal transduction. We have developed an assay based on Fluorescence Resonance Energy Microscopy (FRET) and novel mathematical models to differentiate between clustered and random distributions of fluorophore-bound molecules on the basis of the dependence of FRET intensity on donor and acceptor concentrations. The models are tailored to extended clusters, which may be tightly packed, and account for geometric exclusion effects between membrane-bound proteins. Two main criteria are used to show that labeled polymeric IgA-ligand-receptor complexes are organized in clusters within apical endocytic membranes of polarized MDCK cells: 1), energy transfer efficiency (E%) levels are independent of acceptor levels; and 2), with increasing unquenched donor: acceptor ratio, E% decreases. A quantitative analysis of cluster density indicates that a donor-labeled ligand-receptor complex should have 2.5-3 labeled complexes in its immediate neighborhood and that clustering may occur at a limited number of discrete membrane locations and/or require a specific protein that can be saturated. Here, we present a new sensitive FRET-based method to quantify the co-localization and distribution of ligand-receptor complexes in apical endocytic membranes of polarized cells.     

Cooperative adhesion of ligand-receptor bonds

Cooperative (simultaneous) breakage of multiple adhesive bonds has been proposed as a mechanism for enhanced binding strength between adhesion molecules on apposing cell surfaces. In this report, we used the atomic force microscopy (AFM) to study how changes in binding affinity and separation rate of force-induced ligand-receptor dissociation affect binding cooperativity. The AFM force measurements were carried out using (strept)avidin-functionalized cantilever tips and biotinylated agarose beads under conditions where multiple (strept)avidin-biotin linkages were formed following surface contact. At slow surface separation of the AFM cantilever from the bead's surface, the (strept)avidin-biotin linkages appeared to rupture sequentially. Increasing the separation rate from 210 to 1950 nm/s led to a linear increase in the average rupture force. Moreover, force histograms revealed a quantized force distribution that shifted toward higher values with increasing separation rate. In measurements of streptavidin-iminobiotin adhesion, the force distribution also shifted toward higher values when the buffer was adjusted to a higher pH to raise the binding affinity. Together, these results demonstrate that the cooperativity of ligand-receptor bonds is significantly enhanced by increases in surface separation rate and/or binding affinity.     

Harmonic mean relationship between affinity for wild-type receptors and alanine-scan mutants

Alanine-scanning mutagenesis involves systematic substitution of a single alanine for other amino acid constituents of a ligand or receptor protein. The sequential process generates a discrete set of "mutant" proteins, each member of which differs from the native 'wild-type' receptor (R(wt)) or ligand by the single amino acid (alpha) substitution with alanine (R(Ala)). The Ala mapsto alpha substitutions are made for amino acids suspected of being important for binding of ligand to receptor. Binding assays are then used to measure the affinity for R(wt) and mutants, quantified by the ligand-receptor equilibrium constant (K(eq)) or its reciprocal, the dissociation constant (K(d)=1/K(eq)). However, the relationship betweenR(wt)K(d) and R(Ala)K(d) values is not obvious. Neither the arithmetic mean nor the geometric mean (based on a simplified relationship between K(d) and the thermodynamic standard free energy change) of the R(Ala)K(d) values turns out to be adequate. The harmonic mean was found to be a good estimator when applied to the published data from 14 alanine-scan studies. This result is consonant with the present understanding of ligand-receptor interactions.     

Application of atomic force microscopy for characteristics of single intermolecular interactions

Atomic force microscopy (AFM) can be used to make measurements in vacuum, air, and water. The method is able to gather information about intermolecular interaction forces at the level of single molecules. This review encompasses experimental and theoretical data on the characterization of ligand-receptor interactions by AFM. The advantage of AFM in comparison with other methods developed for the characterization of single molecular interactions is its ability to estimate not only rupture forces, but also thermodynamic and kinetic parameters of the rupture of a complex. The specific features of force spectroscopy applied to ligand-receptor interactions are examined in this review from the stage of the modification of the substrate and the cantilever up to the processing and interpretation of the data. We show the specificities of the statistical analysis of the array of data based on the results of AFM measurements, and we discuss transformation of data into thermodynamic and kinetic parameters (kinetic dissociation constant, Gibbs free energy, enthalpy, and entropy). Particular attention is paid to the study of polyvalent interactions, where the definition of the constants is hampered due to the complex stoichiometry of the reactions.     

The role of flexible tethers in multiple ligand-receptor bond formation between curved surfaces

Ligands mounted to surfaces via extensible tethers are present in nature and represent a growing class of molecules used to engineer adhesion in drug targeting, biosensing, self-assembling nanostructures, and in other biophysical research. Using a continuum approach with geometric and thermodynamic arguments, we derive a number of analytical expressions that relate key properties of single-tethered ligand-receptor interactions to multiple bond formation between curved surfaces. The theoretical predictions are in good agreement with measurements made with the surface forces apparatus. We establish that, when ligated, many tethers commonly used in biophysical research exhibit a discrete binding range that can be accurately measured with force spectroscopy. The distribution of bound ligated tethers is independent of the surfaces' interaction radius, R. The bridging force scales linearly with R, the tether's effective spring constant and grafting density, and with the ligand-receptor bond energy when the surfaces are in direct contact. These results are contrasted to bridging forces that evolve between plane-parallel geometries. Last, we show how our simple analytical reductions can be used to predict adhesive forces for STEALTH liposomes and other targeted and self-assembled nanoparticles.     

An insight into the ligand-receptor interactions involved in the translocation of pathogens across blood-brain barrier

Traversal of pathogen across the blood-brain barrier (BBB) is an essential step for central nervous system (CNS) invasion. Pathogen traversal can occur paracellularly, transcellularly, and/or in infected phagocytes (Trojan horse mechanism). To trigger the translocation processes, mainly through paracellular and transcellular ways, interactions between protein molecules of pathogen and BBB are inevitable. Simply, it takes two to tango: both host receptors and pathogen ligands. Underlying molecular basis of BBB translocation of various pathogens has been revealed in the last decade, and a plethora of experimental data on protein-protein interactions has been created. This review compiles these data and should give insights into the ligand-receptor interactions that occur during BBB translocation. Further, it sheds light on cell signaling events triggered in response to ligand-receptor interaction. Understanding of the molecular principles of pathogen-host interactions that are involved in traversal of the BBB should contribute to develop new vaccine and drug strategies to prevent CNS infections.