Binding Rate Constants Research Articles

Binding kinetics of quaternary ammonium ions in Kcv potassium channels.

Kcv channels from plant viruses represent the autonomous pore module of potassium channels, devoid of any regulatory domains. These small proteins show very reproducible single-channel behavior in planar lipid bilayers. Thus, they are an optimum system for the study of the biophysics of ion transport and gating. Structural models based on homology modeling have been used successfully, but experimental structural data are currently not available. Here we determine the size of the cytosolic pore entrance by studying the blocker kinetics. Blocker binding and dissociation rate constants ranging from 0.01 to 1000 ms-1 were determined for different quaternary ammonium ions. We found that the cytosolic pore entrance of KcvNTS must be at least 11 Å wide. The results further indicate that the residues controlling a cytosolic gate in one of the Kcv isoforms influence blocker binding/dissociation as well as a second gate even when the cytosolic gate is in the open state. The voltage dependence of the rate constant of blocker release is used to test, which blockers bind to the same binding site.

Mechanistic model for epigenetic maintenance by methyl-CpG-binding domain proteins.

DNA methylation is a fundamental element of epigenetic regulation that is governed by the MBD protein superfamily, a group of "readers" that share a highly conserved methyl-CpG-binding domain (MBD) and mediate chromatin remodeler recruitment, transcription regulation, and coordination of DNA and histone modification. Previous work has characterized the binding affinity and sequence selectivity of MBD-containing proteins toward palindromes of 5-methylcytosine (5mC) containing 5mCpG dinucleotides, often referred to as single symmetrically methylated CpG sites. However, little is known about how MBD binding is influenced by the prototypical local clustering of methylated CpG sites and the presence of DNA structural motifs encountered, e.g., during DNA replication and transcription. Here, we use Single-Molecule Kinetics through Equilibrium Poisson Sampling (SiMKEPS) to measure precise binding and dissociation rate constants of the MBD of human protein MBD1 to DNAs with varying patterns of multiple methylated CpG sites and diverse structural motifs. MBD binding is promoted by two major properties of its DNA substrates: 1) tandem (consecutive) symmetrically methylated CpG sites in double-stranded DNA and secondary structures in single-stranded DNA; and 2) DNA forks. Based on our findings, we propose a mechanistic model for how MBD proteins contribute to epigenetic boundary maintenance between transcriptionally silenced and active genome regions.

Studies on the interaction of aquahydroxocobinamide with 5-aminotetrazole

Here we show that 5-aminotetrazole (ATZ) is capable of moderately strong binding with aquahydroxocobinamide ((H2O)(HO-)Cbi). At pH 7.4, 25.0 °C, the reaction of (H2O)(HO-)Cbi with a two-fold excess of ATZ produces a mixture of complexes with one (a major fraction) having two ATZ ligands, and a minor fraction of (H2O)(HO-)Cbi remains unreacted. ATZ complexation with (H2O)(HO-)Cbi proceeds rapidly, i.e. rate constants for binding of the first and second ATZ molecules are (4.3 ± 0.5)×103 and (5.8 ± 0.4)×102 M−1 s−1 (pH 7.4, 25.0 °C), respectively. The addition of ATZ to (H2O)(HO-)Cbi notably decreases the extent of Cbi species complexation with bovine serum albumin and the rate of Cbi(III) species reduction to Cbi(II) by glutathione, whereas the rates of reactions with cyanide and hydrogen sulfide remain relatively high. Thus, ATZ may prevent (H2O)(HO-)Cbi retention by proteins and its involvement in side redox reactions upon introduction in the human body, which explains the high absorption of the Cbi-ATZ complex during intramuscular injection and its high efficacy as an antidote.

Possible interaction between pectin and gluten alters the starch digestibility and texture of wheat bread

This study incorporated citrus pectin in wheat bread, aiming to develop breads with both desirable texture and slow starch digestibility. Results showed that starch digestibility in wheat bread decreased over the addition of pectin, and the maximum starch digested amount decreased by 6.6 % after the addition of 12 % pectin (wheat flour weight basis). The addition of pectin transferred part of the rapidly digestible starch into slowly digestible starch, and reduced the binding rate constant between slowly digestible starch and digestive enzymes, resulting in overall reduced starch digestibility. Furthermore, the addition of 4 % pectin contributed to the development of wheat bread with softer texture and increased specific volume. Mechanistically, the lowered starch digestibility of wheat bread after the pectin addition was due to (1) residual outermost swollen layer of starch granules, (2) protein and pectin interactions, and (3) increased short-range ordering of starch. This study, therefore, suggests that the addition of an appropriate amount of citrus pectin has the potential to develop bread with both a low glycemic index and desirable texture.

A simple method to resolve rate constants when the binding mechanism obeys induced fit or conformational selection

Many interactions involving a ligand and its molecular target are studied by rapid kinetics using a stopped-flow apparatus. Information obtained from these studies is often limited to a single, saturable relaxation that is insufficient to resolve all independent rate constants even for a two-step mechanism of binding obeying induced fit (IF) or conformational selection (CS). We introduce a simple method of general applicability where this limitation is overcome. The method accurately reproduces the rate constants for ligand binding to the serine protease thrombin determined independently from the analysis of multiple relaxations. Application to the inactive zymogen precursor of thrombin, prethrombin-2, resolves all rate constants for a binding mechanism of IF or CS from a single, saturable relaxation. Comparison with thrombin shows that the prethrombin-2 to thrombin conversion enhances ligand binding to the active site not by improving accessibility through the value of kon but by reducing the rate of dissociation koff. The conclusion holds regardless of whether binding is interpreted in terms of IF or CS and has general relevance for the mechanism of zymogen activation of serine proteases. The method also provides a simple test of the validity of IF and CS and indicates when more complex mechanisms of binding should be considered.

Quantifying Imaging Agent Binding and Dissociation in 3-D Cancer Spheroid Tissue Culture Using Paired-Agent Principles.

Binding kinetics play an important role in cancer diagnosis and therapeutics. However, current methods of quantifying binding kinetics fail to consider the three-dimensional environment that drugs and imaging agents experience in biological tissue. In response, a methodology to assay agent binding and dissociation in 3-D tissue culture was developed using paired-agent molecular imaging principles. To test the methodology, the uptakes of ABY-029 (an IRDye 800CW-labeled epidermal growth factor receptor (EGFR)-targeted antibody mimetic) and IRDye-700DX carboxylate in 3-D spheroids were measured in four different human cancer cell lines throughout staining and rinsing. A compartment model (optimized for the application) was then fit to the kinetic curves of both imaging agents to estimate binding and dissociation rate constants of the EGFR-targeted ABY-029 agent. A statistically significant correlation was observed between apparent association rate constant (k3) and the receptor concentration experimentally and in simulations (r = 0.99, p < 0.05). A statistically significant difference was found between effective k3 (apparent rate constant of ABY-029 binding to EGFR) values for cell lines with varying levels of EGFR expression (p < 0.05), with no significant difference found between cell lines and controls for other fit parameters. Additionally, a similar binding affinity profile compared to a gold standard method was determined by this model. This low-cost methodology to quantify imaging agent or drug binding affinity in clinically relevant 3-D tumor spheroid models can be used to guide timing of imaging in molecular guided surgery and could have implications in drug development.

Temperature-Dependent Kinetics for the Reactions of Fen- (n = 2-17) and FexNiy- (x + y = 3-9) with O2: Comparison of Pure and Mixed Metal Clusters with Relevance to Meteor Radio Afterglows and Surface Oxidation.

Rate constants and product branching fractions were measured from 300-600 K for Fen- + O2 (n = 2-17) and for 300-500 K for FexNiy- + O2 (x + y = 3-9) using a selected-ion flow tube (SIFT) apparatus. Rate constants for 46 species are reported. All rate constants increased with increasing temperature, and several were in excess of the Langevin-Gioumousis-Stevenson (LGS) capture rate at elevated temperatures. As with previously studied transition metal anion oxidation reactions, the collision limit is treated as the sum of the LGS limit along with a hard-sphere contribution, allowing for determination of activation energies. These values are compared to each other along with previous results for Nin-. Measured rate constants for all three series (Fen-, Nin-, and FexNy-) vary over a relatively narrow range (1-5 × 10-10 cm3 s-1 at 300 K) being at least 15% of the collision rate constant. All reaction rate constants increase with temperature, described by small activation energies of 0.5-4 kJ mol-1. The data are consistent with an anticorrelation between the electron binding energy and rate constant, previously noted in other systems. The Fen- reaction produces a larger population of higher energy electrons than do the Nin- reactions, with FexNiy- producing an intermediate amount. The results suggest that the overall rate constant is limited by a small energetic barrier located at a large internuclear distance where electrostatic forces dominate, causing the potentials to be similar across systems, while the product formation is determined by the shorter-range, valence portion of the potential, which varies widely between systems.

Revolutionizing the capture efficiency of ultrasensitive digital ELISA via an antibody oriented-immobilization strategy.

Bead-based digital ELISA, the most sensitive protein quantification method, has drawn much attention to exploring ultra-low abundance biomarkers in the life sciences and clinical applications. However, its major challenge refers to the low antigen capture efficiency in the immunoreaction process due to the low probability of collision between the deficient concentration of the analytes and the captured antibody-immobilized on the beads. Here, we achieved significantly improved reaction efficiency in the digital signal formation by fixing the orientation of antibodies and revealed the kinetic mechanism for the first time. A facile and fast antibody conjugation strategy that formed boronate ester complexes was designed to retain the uniform orientation of antibodies with controllable antibody density. Remarkably, the oriented immobilized antibody exhibited stronger antigen-binding capacity and faster antigen-binding speed compared to randomly immobilized antibodies, with capture efficiency increasing approximately 14-fold at 15 μg of antibody per 1 mg microbeads (0.035 antibody nm-2) under 0.5 h incubation. Combined with theoretical analysis, we verified that the improved capture efficiency of the oriented antibodies mainly originated from the considerable rise in the binding rate constant (kon) rather than the increase in antigen-binding sites, which further prominently decreased the limit of detection (LoD) in a shorter incubation time compared with the randomly immobilized antibody. In conclusion, the antibody oriented conjugation method effectively overcomes the low capture efficiency challenge of bead-based digital ELISA. It paves a promising way for further improving the digital immunoassay performance and promotes the early diagnosis of diseases by recognizing more ultra-low abundance significant biomarkers.

SARS-CoV-2S-Protein-Ace2 Binding Analysis Using Surface Plasmon Resonance.

Surface plasmon resonance (SPR) allows for the label-free determination of the binding affinity and rate constants of bimolecular interactions. Here, we describe the method used for the analysis of the Ace2-SARS-CoV2 S-protein interaction using indirect capture of the S-protein onto the SPR surface, and flowing monomeric Ace2. This method will allow for the determination of the rate constants for affinity, with additional analysis that is achievable using S-protein capture levels in conjunction with the sensorgram response for relative activity benchmarking.

In Vitro Methods for the Investigation of sRNA-mRNA Interactions in Bacillus subtilis.

So far, in Bacillus subtilis, only four trans-encoded and 11 cis-encoded sRNAs and their targets have been investigated in detail, the majority of them in our group (rev. in 1, 2). Here, we describe in vitro methods for the analysis of sRNA/mRNA interactions. All these methods have been either elaborated or significantly improved in our group and successfully applied to characterize a number of sRNA/target mRNA systems in Bacillus subtilis for which we provide examples from our own work. The in vitro methods comprise the synthesis and purification of labeled and unlabeled RNA, the analysis of sRNA/mRNA interactions in electrophoretic mobility shift assays (EMSAs) including the calculation of their apparent binding rate constants (kapp) and equilibrium dissociation constants (Kd), the localization of minimal regulatory regions of an sRNA, the determination of the secondary structures of both interacting RNAs and their complex as well as the analysis of RNA chaperones that may promote the sRNA/mRNA interaction.

Elucidating protein-ligand binding kinetics based on returning probability theory.

The returning probability (RP) theory, a rigorous diffusion-influenced reaction theory, enables us to analyze the binding process systematically in terms of thermodynamics and kinetics using molecular dynamics (MD) simulations. Recently, the theory was extended to atomistically describe binding processes by adopting the host-guest interaction energy as the reaction coordinate. The binding rate constants can be estimated by computing the thermodynamic and kinetic properties of the reactive state existing in the binding processes. Here, we propose a methodology based on the RP theory in conjunction with the energy representation theory of solution, applicable to complex binding phenomena, such as protein-ligand binding. The derived scheme of calculating the equilibrium constant between the reactive and dissociate states, required in the RP theory, can be used for arbitrary types of reactive states. We apply the present method to the bindings of small fragment molecules [4-hydroxy-2-butanone (BUT) and methyl methylthiomethyl sulphoxide (DSS)] to FK506 binding protein (FKBP) in an aqueous solution. Estimated binding rate constants are consistent with those obtained from long-timescale MD simulations. Furthermore, by decomposing the rate constants to the thermodynamic and kinetic contributions, we clarify that the higher thermodynamic stability of the reactive state for DSS causes the faster binding kinetics compared with BUT.

Kinetics of Oxygen Coordination to a Graphite Supported Cobalt Porphyrin

Single molecule level time-dependent binding of O2 to cobalt(II) octaethylporphyrin [CoOEP] supported on graphite at the solution-solid interface was observed with scanning tunneling microscopy (STM). Using STM movies of the surface and a stochastic kinetic analysis it was possible to extract both the binding and dissociation rate constants for this reversible equilibrium process. This study was the first of its kind and has opened the way for kinetic analysis if reversible surface reactions at the single molecule level by STM methodology. These results are transformative for STM based studies because they demonstrate both the possibility of, and the methodology for, performing kinetic binding studies at the solution solid interface at room temperature.

Nitric oxide binding to ferrous nitrobindins: A computer simulation investigation

Nitrobindins (Nbs) represent an evolutionary conserved all-β-barrel heme-proteins displaying a highly solvent-exposed heme-Fe(III) atom, coordinated by a proximal His residue. Interestingly, even if the distal side is exposed to the solvent, the value of the second order rate constants for ligand binding to the ferrous derivative is almost one order of magnitude lower than those reported for myoglobins (Mbs). Noteworthy, nitric oxide binding to the sixth coordination position of the heme-Fe(II)-atom causes the cleavage or the severe weakening of the proximal His-Fe(II) bond. Here, we provide a computer simulation investigation to shed light on the molecular basis of ligand binding kinetics, by dissecting the ligand binding process into the ligand migration and the bond formation steps. Classical molecular dynamics simulations were performed employing a steered molecular dynamics approach and the Jarzinski equality to obtain ligand migration free energy profiles. The formation of the heme-Fe(II)-NO bond took into consideration the iron atom displacement from the heme plane. The ligand migration is almost unhindered, and the low rate constant for NO binding is due to the large displacement of the Fe(II) atom with respect to the heme plane responsible for the barrier for the Fe(II)-NO bond formation. In addition, we investigated the weakening and breaking of the proximal His-Fe(II) bond, observed experimentally upon NO binding, by means of a combination of classical molecular dynamics simulations and quantum–classical (QM-MM) optimizations. In both human and M. tuberculosis Nbs, a stable alternative conformation of the proximal His residue interacting with a network of water molecules was observed.

Binding of Nitriles and Isonitriles to V(III) and Mo(III) Complexes: Ligand vs Metal Controlled Mechanism.

The synthesis and structures of nitrile complexes of V(N[tBu]Ar)3, 2 (Ar = 3,5-Me2C6H3), are described. Thermochemical and kinetic data for their formation were determined by variable temperature Fourier transform infrared (FTIR), calorimetry, and stopped-flow techniques. The extent of back-bonding from metal to coordinated nitrile indicates that electron donation from the metal to the nitrile plays a less prominent role for 2 than for the related complex Mo(N[tBu]Ar)3, 1. Kinetic studies reveal similar rate constants for nitrile binding to 2, but the activation parameters depend critically on the nature of R in RCN. Activation enthalpies range from 2.9 to 7.2 kcal·mol-1, and activation entropies from -9 to -28 cal·mol-1·K-1 in an opposing manner. Density functional theory (DFT) calculations provide a plausible explanation supporting the formation of a π-stacking interaction between a pendant arene of the metal anilide of 2 and the arene substituent on the incoming nitrile in favorable cases. Data for ligand binding to 1 do not exhibit this range of activation parameters and are clustered in a small area centered at ΔH‡ = 5.0 kcal·mol-1 and ΔS‡ = -26 cal·mol-1·K-1. Computational studies are in agreement with the experimental data and indicate a stronger dependence on electronic factors associated with the change in spin state upon ligand binding to 1.

SARS-CoV-2 Binding to Terminal Sialic Acid of Gangliosides Embedded in Lipid Membranes.

Multiple recent reports indicate that the S protein of SARS-CoV-2 specifically interacts with membrane receptors and attachment factors other than ACE2. They likely have an active role in cellular attachment and entry of the virus. In this article, we examined the binding of SARS-CoV-2 particles to gangliosides embedded in supported lipid bilayers (SLBs), mimicking the cell membrane-like environment. We show that the virus specifically binds to sialylated (sialic acid (SIA)) gangliosides, i.e., GD1a, GM3, and GM1, as determined from the acquired single-particle fluorescence images using a time-lapse total internal reflection fluorescence (TIRF) microscope. The data of virus binding events, the apparent binding rate constant, and the maximum virus coverage on the ganglioside-rich SLBs show that the virus particles have a higher binding affinity toward the GD1a and GM3 compared to the GM1 ganglioside. Enzymatic hydrolysis of the SIA-Gal bond of the gangliosides confirms that the SIA sugar unit of GD1a and GM3 is essential for virus attachment to the SLBs and even the cell surface sialic acid is critical for the cellular attachment of the virus. The structural difference between GM3/GD1a and GM1 is the presence of SIA at the main or branched chain. We conclude that the number of SIA per ganglioside can weakly influence the initial binding rate of SARS-CoV-2 particles, whereas the terminal or more exposed SIA is critical for the virus binding to the gangliosides in SLBs.

Effect of trifluoroethanol on antibodies binding properties

The studies on the influence of organic co-solvents on the structure and function of antibodies are of key interest, especially in view of antibodies broad use as recognizing elements in different analytical systems. Here we studied the effect of co-solvent 2,2,2-trifluoroethanol (TFE) on the ability of anti-ovalbumin monoclonal antibodies to interact with its specific antigen. Antibody affinity to antigen and the rate constants of antibody binding to immobilized antigen were analyzed. Changes in antibody reactivity with incubation time which depended on TFE concentration and temperature were revealed. When treatment of antibodies with TFE was carried out at 0°C, we observed nonlinear, non-monotonous changes of antibody reactivity with initial fast decrease and substantial increase that may be related to the loss of antigen binding reactivity by some part of antibodies at the start but its restoration when the incubation proceeds. Keywords: 2;2;2-trifluoroethanol, antibody affinity, antigen-antibody interaction, monoclonal antibodies, ovalbumin

Dominance analysis of competing protein assembly pathways.

Most proteins form complexes consisting of two or more subunits, where complex assembly can proceed via two competing pathways: co-translational assembly of a mature and a nascent subunit, and post-translational assembly by two mature protein subunits. Assembly pathway dominance, i.e., which of the two pathways is predominant under which conditions, is poorly understood. Here, we introduce a reaction-diffusion system that describes protein complex formation via post- and co-translational assembly and use it to analyze the dominance of both pathways. Special features of this new system are (i) spatially inhomogeneous sources of reacting species, (ii) a combination of diffusing and immobile species, and (iii) an asymmetric binding competition between the species. We study assembly pathway dominance for the spatially homogeneous system and find that the ratio of production rates of the two protein subunits determines the long-term pathway dominance. This result is independent of the binding rate constants for post- and co-translational assembly and implies that a system with an initial post-translational assembly dominance can eventually exhibit co-translational assembly dominance and vice versa. For exactly balanced production of both subunits, the assembly pathway dominance is determined by the steady state concentration of the subunit that can bind both nascent and mature partners. The introduced system of equations can be applied to describe general dynamics of assembly processes involving both diffusing and immobile components.

Structural basis for rice starch multi-digestible fractions revealed by consecutive reaction kinetics model.

Starch-based foods (e.g. rice) usually contain multiple starch fractions with distinct digestion rate constants, although their nature is currently unknown. The present study applied the recently developed consecutive reaction kinetics model to fit the in vitro digestion curves for starch fractions deconvoluted from the overall digestograms to differentiate their binding and catalysis rates to starch digestive enzymes. The fitting parameters were then correlated with starch molecular structures obtained from published data to understand starch structural features determining the binding and catalytic rate constants. Binding and catalysis rates for the rapidly (RDF) and slowly digestible starch fraction (SDF) were controlled by distinct starch structural features. Typically, (i) the binding rate constant for RDF was negatively correlated with the amount of amylose short to intermediate chains, whereas it was positively correlated with the relative length of amylopectin intermediate chains; (ii) the catalysis rate constant for RDF was negatively correlated with the amount of amylose short to intermediate chains, relative length of amylose intermediate chains and amount of amylopectin long chains, whereas it was positively correlated with starch molecular size as well as relative length of amylopectin intermediate chains; (iii) and the catalysis rate constant for SDF was negatively correlated with the amount of amylopectin long chains, whereas it was positively correlated with starch molecular size. These results provide a better understanding of the nature of different starch digestible fractions and the development of foods such as rice with slow starch digestibility. © 2023 Society of Chemical Industry.

Incorporation of rapid association/dissociation processes in tissues into the monkey and human physiologically based pharmacokinetic models for manganese.

In earlier physiologically based pharmacokinetic (PBPK) models for manganese (Mn), the kinetics of transport of Mn into and out of tissues were primarily driven by slow rates of association and dissociation of Mn with tissue binding sites. However, Mn is known to show rapidly reversible binding in tissues. An updated Mn model for primates, following similar work with rats, was developed that included rapid association/dissociation processes with tissue Mn-binding sites, accumulation of free Mn in tissues after saturation of these Mn-binding sites and rapid rates of entry into tissues. This alternative structure successfully described Mn kinetics in tissues in monkeys exposed to Mn via various routes including oral, inhalation, and intraperitoneal, subcutaneous, or intravenous injection and whole-body kinetics and tissue levels in humans. An important contribution of this effort is showing that the extension of the rate constants for binding and cellular uptake established in the monkey were also able to describe kinetic data from humans. With a consistent model structure for monkeys and humans, there is less need to rely on cadaver data and whole-body tracer studies alone to calibrate a human model. The increased biological relevance of the Mn model structure and parameters provides greater confidence in applying the Mn PBPK models to risk assessment. This model is also well-suited to explicitly incorporate emerging information on the role of transporters in tissue disposition, intestinal uptake, and hepatobiliary excretion of Mn.

Conventional Receptor Radioligand Binding Techniques Applied to the Study of Monoamine Oxidase.

Designed to measure binding interactions between small molecules and receptor proteins, radioligand binding approaches may also be applied to interactions between monoamine oxidase (MAO) and its ligands. The technique may be used with tissue homogenates or with mitochondrial membranes and can provide information about binding site density, ligand affinity, binding rate constants, and binding events at sites that do not impact absorbance characteristics of the flavin cofactor and that may not be amenable to spectrophotometric studies. This overview describes the use of a cell harvester in a common filtration approach to measure binding to MAO of radiolabeled substrates, inhibitors, or allosteric ligands in saturation analyses and to take advantage of the principles of competition to obtain quantitative binding data for unlabeled ligands that may bind with much lower affinity. The quality and reproducibility of data are impacted by factors such as choice of ligand concentrations, pipetting technique, graphing and regression approaches, and scintillation counting parameters, and consideration is given to these and other factors that may influence the results.