scholarly journals The structure of binding curves and practical identifiability of equilibrium ligand-binding parameters

2016 ◽  
Vol 149 (1) ◽  
pp. 121-147 ◽  
Author(s):  
Thomas R. Middendorf ◽  
Richard W. Aldrich
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Association Constants ◽  
Binding Parameters ◽  
Parameter Identifiability ◽  
Binding Parameter ◽  
Binding Data ◽  
Binding Curve ◽  
Site Binding ◽  
Binding Curves

A critical but often overlooked question in the study of ligands binding to proteins is whether the parameters obtained from analyzing binding data are practically identifiable (PI), i.e., whether the estimates obtained from fitting models to noisy data are accurate and unique. Here we report a general approach to assess and understand binding parameter identifiability, which provides a toolkit to assist experimentalists in the design of binding studies and in the analysis of binding data. The partial fraction (PF) expansion technique is used to decompose binding curves for proteins with n ligand-binding sites exactly and uniquely into n components, each of which has the form of a one-site binding curve. The association constants of the PF component curves, being the roots of an n-th order polynomial, may be real or complex. We demonstrate a fundamental connection between binding parameter identifiability and the nature of these one-site association constants: all binding parameters are identifiable if the constants are all real and distinct; otherwise, at least some of the parameters are not identifiable. The theory is used to construct identifiability maps from which the practical identifiability of binding parameters for any two-, three-, or four-site binding curve can be assessed. Instructions for extending the method to generate identifiability maps for proteins with more than four binding sites are also given. Further analysis of the identifiability maps leads to the simple rule that the maximum number of structurally identifiable binding parameters (shown in the previous paper to be equal to n) will also be PI only if the binding curve line shape contains n resolved components.

Analysis of experimental binding curves of EtBr with single- and double-stranded DNA at small fillings

2014 ◽  
Vol 28 (22) ◽  
pp. 1450178 ◽  
Author(s):  
Poghos O. Vardevanyan ◽  
Valeri B. Arakelyan ◽  
Marine A. Parsadanyan ◽  
Ara P. Antonyan ◽  
Gohar G. Hovhannisyan ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Binding Mode ◽  
Ligand Molecule ◽  
Binding Parameters ◽  
Base Pairs ◽  
Precise Data ◽  
Double Stranded Dna ◽  
Single Curve ◽  
Binding Curves

In this paper, a method that allows to analyze the binding curves of ligand ( EtBr ) with single-stranded (ss) and double-stranded (ds) DNA, when there are at least two modes of ligand binding to DNA at small fillings has been proposed. The obtained experimental binding curves for EtBr –ssDNA and EtBr –dsDNA have two clearly expressed linear regions. These curves were analyzed by two modes: Experimental points on linear regions were described by two different lines and all experimental points were described by single curve. It was revealed that the description by single curve permits obtaining more precise data of binding parameters (i.e. binding constant and number of base pairs that bind one ligand molecule). Moreover, the proposed method permits determining the value of proportion of binding sites of each binding mode.

Interaction of Ca++ binding sites of troponin: significance for Ca++ movements

1975 ◽  
Vol 228 (1) ◽  
pp. 172-178 ◽  
Author(s):  
CR Honig ◽  
YS Reddy
Keyword(s):  
Muscle Contraction ◽  
Least Squares ◽  
Binding Sites ◽  
Time Course ◽  
Nonlinear Least Squares ◽  
Cooperative Effect ◽  
Active State ◽  
Binding Parameters ◽  
Binding Data ◽  
Intracellular Ca

Ca++ binding data for seven preparations of troponin and 37 preparations of native tropomyosin were analyzed by the method of nonlinear least squares. The analysis was based on the assumption that two classes of independent binding sites exist. Data from one-third the preparations could not be fitted with all binding parameters at true least-squares minimum, and computed values of parameters for two-thirds the preparations were biologically uniterpretable. We conclude that troponin does not contain two classes of independent binding sites. Comparison of Scratchard plots of Ca++ binding by troponin and native tropomyosin modifies binding through a cooperative effect on troponin. Certain features of the Scratchard plots are imcompatible with the possibility that troponin possess more than two classes of independent sites. We interpret these results to mean that troponin's binding sites interact and that the interaction is increased by tropomyosin.The interaction would cause Ca++ affinity to vary with time during a muscle contraction. The effect of variable Ca++ affinity on intracellular Ca++ movements and the time course of the active state is discussed.

scholarly journals Free-energy relationships in ion channels activated by voltage and ligand

2012 ◽  
Vol 141 (1) ◽  
pp. 11-28 ◽  
Author(s):  
Sandipan Chowdhury ◽  
Baron Chanda
Keyword(s):  
Free Energy ◽  
Ion Channels ◽  
Ligand Binding ◽  
Interaction Energy ◽  
Bk Channel ◽  
Direct Estimate ◽  
Voltage Dependent ◽  
Energy Relationships ◽  
Binding Curve ◽  
Binding Curves

Many ion channels are modulated by multiple stimuli, which allow them to integrate a variety of cellular signals and precisely respond to physiological needs. Understanding how these different signaling pathways interact has been a challenge in part because of the complexity of underlying models. In this study, we analyzed the energetic relationships in polymodal ion channels using linkage principles. We first show that in proteins dually modulated by voltage and ligand, the net free-energy change can be obtained by measuring the charge-voltage (Q-V) relationship in zero ligand condition and the ligand binding curve at highly depolarizing membrane voltages. Next, we show that the voltage-dependent changes in ligand occupancy of the protein can be directly obtained by measuring the Q-V curves at multiple ligand concentrations. When a single reference ligand binding curve is available, this relationship allows us to reconstruct ligand binding curves at different voltages. More significantly, we establish that the shift of the Q-V curve between zero and saturating ligand concentration is a direct estimate of the interaction energy between the ligand- and voltage-dependent pathway. These free-energy relationships were tested by numerical simulations of a detailed gating model of the BK channel. Furthermore, as a proof of principle, we estimate the interaction energy between the ligand binding and voltage-dependent pathways for HCN2 channels whose ligand binding curves at various voltages are available. These emerging principles will be useful for high-throughput mutagenesis studies aimed at identifying interaction pathways between various regulatory domains in a polymodal ion channel.

scholarly journals Biophysical cytochemical investigations of intracellular heparin in neoplastic mast cells.

1980 ◽  
Vol 28 (3) ◽  
pp. 223-230 ◽  
Author(s):  
N Nakamura ◽  
R E Hurst ◽  
S S West ◽  
J M Menter ◽  
J F Golden ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Binding Constant ◽  
Binding Constants ◽  
Cooperative Binding ◽  
Binding Parameters ◽  
Binding Studies ◽  
Mastocytoma Cells ◽  
Biochemical Analyses

The thermodynamic binding parameters for intracellular heparin-acridine orange (AO) complexes were determined for Furth murine mastocytoma cells and were found to agree with 1) results from binding studies on heparin-AO complexes in solution, and 2) with biochemical analyses of the cells. The cells exhibited cooperative binding with a binding constant of 1.18 x 10(6) M-1. The cooperative binding constant of heparin-AO in 1 mM buffer was found to be 1.13 x 10(6) M-1. The addition of 1 mM NaCl to heparin-AO system in vitro detectably decreased the cooperative binding constant. Low ionic strength is the only condition in solution under which the cell and solution binding constants are equal. The cells have an average of 1.2 x 10(-14) mol of AO binding sites per cell. Using the biochemically measured heparin content per cell and the amount of AO bound by heparin in solution, 8 x 10(15) mol of sites/cell can be attributed to heparin. The remaining cellular binding sites (4 x 10(-15) mol of sites per cell) are essentially all accounted for by AO binding to DNA, the amount of which is calculated from its previously determined thermodynamic binding parameters. A theoretical isotherm, calculated from the binding parameters of both heparin-AO in solution and DNA-AO complexes in situ, agreed closely with the isotherm experimentally determined for the Furth mastocytoma cells. Ligand-binding analysis yields a binding constant, which may aid in identification of cellular bipolymers, and the number of ligand binding sites per cell. The latter is a measure of the amount of a given intracellular biopolymer present.

scholarly journals Structural basis of negative cooperativity in transthyretin.

2001 ◽  
Vol 48 (4) ◽  
pp. 867-875 ◽  
Author(s):  
P Neumann ◽  
V Cody ◽  
A Wojtczak
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Structural Basis ◽  
Negative Cooperativity ◽  
Channel Diameter ◽  
Interatomic Distances ◽  
Sequence Of Events ◽  
Different Sources ◽  
Site Binding ◽  
Made In

A comparison of the AC and BD binding sites of transthyretin (TTR) was made in terms of the interatomic distances between the Ca atoms of equivalent amino acids, measured across the tetramer channel in each binding site. The comparison of the channel diameter for apo TTR from different sources revealed that in the unliganded transthyretin tetramers the distances between the A, D and H beta-strands are consistently larger, while the distances between the G beta-strands are smaller in one site than in the other. These differences might be described to have a 'wave' character. An analogous analysis performed for transthyretin complexes reveals that the shape of the plot is similar, although the amplitudes of the changes are smaller. The analysis leads us to a model of the changes in the binding sites caused by ligand binding. The sequence of events includes ligand binding in the first site, followed by a slight collapse of this site and concomitant opening of the second site, binding of the second molecule and collapse of the second site. The following opening of the first, already occupied site upon ligand binding in the second site is smaller because of the bridging interactions already formed by the first ligand. This explains the negative cooperativity (NC) effect observed for many ligands in transthyretin.

scholarly journals Negative co-operativity in the EGF receptor

2012 ◽  
Vol 40 (1) ◽  
pp. 15-19 ◽  
Author(s):  
Linda J. Pike
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Egf Receptor ◽  
The Other ◽  
New Approach ◽  
X Ray ◽  
High Affinity ◽  
Binding Data ◽  
Epidermal Growth ◽  
Scatchard Plots

Scatchard analyses of the binding of EGF (epidermal growth factor) to its receptor (EGFR) yield concave up Scatchard plots, indicative of some type of heterogenity in ligand-binding affinity. This was typically interpreted as being due to the presence of two independent binding sites: one of high affinity representing ≤10% of the receptor population, and one of low affinity making up the bulk of the receptors. However, the concept of two independent binding sites is difficult to reconcile with the X-ray structures of the dimerized EGFR that show symmetrical binding of the two ligands. A new approach to the analysis of 125I-EGF-binding data combined with the structure of the singly-occupied Drosophila EGFR have now shown that this heterogeneity is due to the presence of negative co-operativity in the EGFR. Concerns that negative co-operativity precludes ligand-induced dimerization of the EGFR confuse the concepts of linkage and co-operativity. Linkage refers to the effect of ligand on the assembly of dimers, whereas co-operativity refers to the effect of ligand binding to one subunit on ligand binding to the other subunit within a preassembled dimer. Binding of EGF to its receptor is positively linked with dimer assembly, but shows negative co-operativity within the dimer.

Albumin/calcium association at different pH, as determined by potentiometry.

1977 ◽  
Vol 23 (11) ◽  
pp. 2122-2126 ◽  
Author(s):  
N Fogh-Andersen
Keyword(s):  
Ionic Strength ◽  
Binding Sites ◽  
Calcium Binding ◽  
Association Constants ◽  
Free Calcium ◽  
Substance Concentration ◽  
Binding Data

Abstract Calcium binding by albumin was determined potentiometrically at physiological ionic strength and temperature as a function of pH. The binding data indicate at least 30 different binding sites with different association constants and different H+ interaction. One site appears to be responsible for the major binding at physiological pH and substance concentration of free calcium, together with three other sites that bind with less affinity.

scholarly journals Binding of α-thrombin to fibrin depends on the quality of the fibrin network

1994 ◽  
Vol 298 (1) ◽  
pp. 157-163 ◽  
Author(s):  
H Bänninger ◽  
B Lämmle ◽  
M Furlan
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Scatchard Analysis ◽  
Binding Parameters ◽  
Irreversible Binding ◽  
Binding Data ◽  
Fibrin Network ◽  
Straight Lines ◽  
Fold Excess

Binding of human alpha-thrombin to fibrin was studied in a purified system at pH 7.35, I 0.08 and 37 degrees C. Binding experiments with active thrombin resulted in fibrin clots of variable quality, depending on the thrombin concentration: opaque gels composed of ‘coarse’ network were produced at low thrombin concentrations, while increasing concentrations of thrombin led to more translucent ‘fine’ gels. Scatchard analysis showed a non-linear dependence of thrombin binding to fibrin, suggesting the existence in fibrin(ogen) of multiple classes of binding sites for thrombin. Binding of catalytic-site-inhibited thrombin was investigated in clots of defined quality produced with three different concentrations of a thrombin-like enzyme, batroxobin (EC 3.4.21.29). Straight lines of different slopes were established by Scatchard analysis of binding data at each fixed batroxobin concentration. These results favour a model according to which binding affinity for thrombin depends on the thickness of fibrin bundles. Labelled active-site-inactivated thrombin incorporated in batroxobin-induced clots was only sparingly released during incubation for 24 h in the presence of a 200-fold excess of unlabelled thrombin, indicating that thrombin binding to fibrin is not reversible and that Scatchard analysis is not appropriate for quantification of binding parameters. Irreversible binding of thrombin appears to reflect trapping of thrombin molecules within fibrin fibres. The amount of trapped thrombin depends on the quality of the fibrin fibres, which in turn is determined by the concentration of the clotting enzyme.

Abnormal Platelet Serotonin Uptake and Binding Sites in Myeloproliferative Disorders

1984 ◽  
Vol 51 (03) ◽  
pp. 349-353 ◽  
Author(s):  
C Caranobe ◽  
P Sié ◽  
F Fernandez ◽  
J Pris ◽  
S Moatti ◽  
...  
Keyword(s):  
Binding Sites ◽  
High Capacity ◽  
Specific Inhibitor ◽  
Myeloproliferative Disorders ◽  
Normal Subjects ◽  
Binding Data ◽  
Full Explanation ◽  
Number Of Binding Sites ◽  
5 Ht Uptake ◽  
Kinetics Of

SummaryA simultaneous investigation of the kinetics of serotonin (5 HT) uptake and of binding sites was carried out in the platelets of normal subjects and of 10 patients affected with various types of myeloproliferative disorders (MD). The 5 HT uptake was analysed according to the Lineweaver-Burk and the Eadie-Hofstee methods. With the two methods, the patient’s platelets exhibited a dramatic reduction of the Vi max and of the Km; in some patients the Eadie-Hofstee analysis revealed that a passive diffusion phenomenon is superimposed on the active 5 HT uptake at least for the higher concentration used. The binding data were analysed with the Scatchard method. Two classes of binding sites (high affinity - low capacity, low affinity - high capacity) were found in normal subjects and patients. Pharmacological studies with imipramine, a specific inhibitor of 5 HT uptake, suggested that both the sites are involved in 5 HT uptake. The number of both binding sites was significantly decreased in patient’s platelets while the affinity constants of these binding sites were not significantly reduced in comparison with those of the control subjects. No correlations were found between Vi max, Km and the number of binding sites. These results suggest that a reduction in the number of platelet membrane acceptors for 5 HT commonly occurs in myeloproliferative disorders but does not provide a full explanation of the uptake defect.

Reversibly Sampling Conformations and Binding Modes Using Molecular Darting

2020 ◽  
Author(s):  
Samuel C. Gill ◽  
David Mobley
Keyword(s):  
Monte Carlo ◽  
Ligand Binding ◽  
Binding Sites ◽  
Degrees Of Freedom ◽  
Dynamics Simulation ◽  
Hiv Integrase ◽  
Binding Modes ◽  
System A ◽  
Multiple Binding ◽  
Internal Degrees Of Freedom

<div>Sampling multiple binding modes of a ligand in a single molecular dynamics simulation is difficult. A given ligand may have many internal degrees of freedom, along with many different ways it might orient itself a binding site or across several binding sites, all of which might be separated by large energy barriers. We have developed a novel Monte Carlo move called Molecular Darting (MolDarting) to reversibly sample between predefined binding modes of a ligand. Here, we couple this with nonequilibrium candidate Monte Carlo (NCMC) to improve acceptance of moves.</div><div>We apply this technique to a simple dipeptide system, a ligand binding to T4 Lysozyme L99A, and ligand binding to HIV integrase in order to test this new method. We observe significant increases in acceptance compared to uniformly sampling the internal, and rotational/translational degrees of freedom in these systems.</div>

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