Some possible peculiarities of a ligand molecule binding inside of a protein macromolecule

Cytochrome P450 3A4 (CYP3A4) metabolizes more than 50% of clinically used drugs and is often involved in adverse drug-drug interactions. It displays atypical binding and kinetic behavior toward a number of ligands characterized by a sigmoidal shape of the corresponding titration curves, which is indicative of a positive homotropic cooperativity. This requires a participation of at least two ligand molecules, whereby the binding of the first ligand molecule increases the affinity of CYP3A4 for the binding of the second ligand molecule. In the current study, a combination of molecular dynamics simulations and free-energy calculations was applied to elucidate the physicochemical origin of the observed positive homotropic cooperativity in ketoconazole binding to CYP3A4. The binding of the first ketoconazole molecule was established to increase the affinity for the binding of the second ketoconazole molecule by 5 kJ mol(-1), which explains and quantifies the experimentally observed cooperative behavior of CYP3A4. Shape complementarity through nonpolar van der Waals interactions was identified as the main driving force of this binding, which seems to be in line with the promiscuous nature of CYP3A4. Moreover, the calculated binding free energies were found to be in good agreement with the values predicted from a simple 2-ligand binding kinetic model as well as to successfully reproduce the experimental titration curve. This confirms the general applicability of rapid free-energy methods to study challenging biomolecular systems like cytochromes P450, which are characterized by a large flexibility and malleability of their active sites.

The kinetics of oxygen binding to human hemoglobin at pH 7, 0.1 m phosphate, 20°, 2.5 x 10-5 to 10-3 m heme has been investigated by chemical relaxation methods in order to obtain information about the elementary steps and the mechanism of the cooperative process of ligand binding. The temperature jump relaxation method allows the oxygen-binding reaction to be followed over its entire time range. The relaxation process is characterized by two phases which are well separated in time. Both the time constants and the relative amplitudes are dependent on protein and ligand concentration. The two relaxation phases can be understood qualitatively. The fast phase corresponds at low oxygen saturation mainly to the binding of the first ligand molecule and at high saturation mainly to the binding of the last ligand molecule. At intermediate oxygen saturation the fast phase is determined predominantly by the kinetics of both the first and the last step—the intermediate reaction steps do not contribute significantly. The slow relaxation phase involves all elementary binding reactions, but is determined mainly by the kinetics of the intermediate oxygen-binding steps. The relative amplitudes of the fast and the slow relaxation phases reflect strongly the population of the reaction species involved. It is concluded that hemoglobin species with one and three ligands bound must be populated measurably at equilibrium. Reaction models which assume negligible concentrations of these intermediates are therefore not valid. Three reactions have been characterized directly by their kinetic properties: (a) a fast reaction step at low oxygen saturation yielding an apparent off-rate constant of about 1000 s-1 and an apparent on-rate constant (per tetramer) of about 4 x 107 m-1 s-1; (b) a fast reaction step at high oxygen saturation, yielding an apparent on-rate constant of about 4 x 107 m-1 s-1; (c) a slow reaction, which at high oxygen concentration yields an apparent rate constant of 5 x 106 m-1 s-1. It thus appears that the binding of the first oxygen molecule to deoxyhemoglobin is a very rapid process which is characterized by an apparent rate constant as large as that found for the binding of the last oxygen molecule. The low oxygen affinity of deoxyhemoglobin arises mainly from the high dissociation rate constant, i.e. short lifetime, of Hb4O2. It cannot be decided if these kinetic properties apply equally to both the α and β chains or if only one type of chain is involved in the fast phase of the relaxation spectrum. A four-step binding model (Adair scheme) explains quantitatively the principal observations of the relaxation kinetics. Adair rate parameters have been obtained which describe the appearance of two relaxation phases only, the ligand concentration dependence (from 0 to 250 µm O2), the protein concentration dependence (from 2 x 10-5 to 10-3 m heme), and the relative and absolute relaxation amplitudes of both phases. The calculated stopped flow kinetic progress curve, using these values, is in agreement with the available data (which are in the saturation range greater than 50%) and predicts a biphasic time course, in contrast to the kinetics of CO-binding. A decision between mechanisms based upon the relaxation kinetics data can be made only with certain assumptions. The values of the Adair-recombination rate constants exclude a mechanism in which there are two very rapidly interconverting structure forms of the protein, each exhibiting equal or nearly equal intrinsic kinetic and equilibrium properties for the four subunits. A concerted mechanism might apply if structure changes are not very fast compared to ligand binding or, if the kinetic properties of the α and β chains are markedly different. However, no consistent fit of the relaxation spectrum has been obtained so far with these assumptions.

Ca2+-binding to proteins can be measured directly by equilibrium dialysis (,), the standard method for the direct measurement of the binding of small ligand molecules by macromolecules. In this method, a semipermeable cellulose bag containing a solution of macromolecules is immersed in the buffer solution containing ligand molecules and is incubated to attain both the chemical and diffusion equilibrium. The method can be improved with the use of two small thin chambers separated by the cellulose membrane, which may reduce the incubation time required to achieve diffusion equilibrium (microdialysis) (). Ligand molecules are usually labeled with the radioactive isotopes for quantitative determinations, and ligand molecules bound to the macromolecule in the equilibrium state are determined directly from the difference between the free concentration in the dialysate and the total concentration in the protein solution. Binding of ligand to the protein molecule can be calculated from the known value of the protein concentration, and the ligand bindings at several free concentrations of the ligand are determined from independent experiments to yield a ligand binding curve from which the maximum number of ligand binding and the equilibrium constants are estimated. In this method, the ligand binding equilibrium, which is usually obtained within less than a second, has to be assessed after attainment of the diffusion equilibrium of ligands across the membrane, which usually takes a much longer time - on the order of several hours.

PfHGXPRT is a key enzyme involved in purine nucleotide salvage pathway of the malarial parasite, Plasmodium falciparum. Atomistic molecular dynamics simulations have been performed on two types of PfHGXPRT dimers (D1 and D3) and its tetramer in their apo and ligand-bound states. A significant event in the catalytic cycle is the dynamics of a gate that provides access for the ligand molecules to the reaction center. The gate is formed by loops II and IV, the former being the most flexible. Large amplitude conformational changes have been observed in active site loop II. Upon complete occupancy of the active site, loop II gets stabilized due to specific interactions between its residues and the ligand molecules. Remote loop, X, is seen to be less fluxional in the D3 dimer than in D1 which is rationalized as due to the greater number of inter-subunit contacts in the former. The presence of ligand molecules in subunits of the tetramer further reduces the flexibility of loop X epitomizing a communication between this region and the active sites in the tetramer. These observations are in accordance with the outcomes of several experimental investigations. Participation of loop X in the oligomerization process has also been discerned. Between the two types of dimers in solution, D1 tetramerizes readily and thus would not be present as free dimers. We conjecture an equilibrium to exist between D3 and the tetramer in solution; upon binding of the ligand molecules to the D3 dimer, this equilibrium shifts toward the tetramer.

A cryogenic technique for the isolation of the ligation intermediates in the association reaction between hemoglobin and carbon monoxide at 20 degrees C [Perrella, M., Davids, N., and Rossi-Bernardi, L. (1992) J. Biol. Chem. 267, 8744-8751] was used to study the effects of proton and chloride concentrations on the rates of the stepwise reactions. The reaction rate was observed to increase continuously in the course of the ligation process, yet the acceleration of the reaction after the binding of two ligand molecules, observed previously in 100 mM KCl, pH 7, was not observed at other pH values. At pH 6.3, such an acceleration occurred after the binding of three ligands, and at pH 8.5, a large acceleration was observed after the binding of the first ligand molecule. Greater CO binding to the beta chains was observed under all conditions, as in the previous study. The functional heterogeneity of the chains in the first ligation step increased with pH. The chloride concentration did not influence the distribution of the ligand between the alpha and beta chains at pH 6.3 and 8.5. At pH 7, less binding to the alpha chains was observed at 7 mM chloride with respect to 100 mM. The nature of the biliganded component isolated at pH 7 in 100 mM KCl and unresolved by the cryogenic technique was studied using a combination of cryogenic and noncryogenic isoelectric focusing. This component was a mixture of intermediates (alpha beta) (alpha CO beta CO), about 65%, and (alpha beta CO) (alpha CO beta), about 35%. The experimental data were compared with the distributions of intermediates calculated according to the Monod kinetic model assuming rapid and concerted transitions between two quaternary structures at each ligation step. The model provided a qualitative fit of the observed distributions of intermediates at acidic and neutral pH. A large discrepancy between the experimental observations and the predictions of the model was found at alkaline pH. The mechanism of the association reaction is discussed in the light of the available information on the tertiary/quaternary structures of the intermediates, as obtained from the studies of the deoxy/cyanomet model of ligation.

Changes in the structure and sizes of human and bovine serum albumins as well as polyvinyl alcohol macromolecules in aqueous solutions depending on temperature, concentration, and acid-base balance (pH) of the solutions are discussed. It is taken into consideration that the change in the hydrodynamic radius of a macromolecule is one of the indicators of structural phase transformations of globular proteins. The methods of the macromolecular radii determination from the shear viscosity and the self-diffusion of macromolecules in solutions are discussed. The hydrodynamic radius values of albumin and polyvinyl alcohol macromolecules obtained by the above methods are thoroughly compared. Consideration of these questions provides us with important information on the nature of the binding of water molecules with protein macromolecules.

Structural Insight into the Constitutive Repression Function of the Nuclear Receptor Rev-erbβ

Proton nuclear magnetic resonance spectroscopy has been used to characterise and compare wild-type fungal and recombinant Coprinus cinereus peroxidase (CIP) and three mutants in which Gly156 and/or Asn157 was replaced by Phe. Analysis of one- and two-dimensional NMR spectra of recombinant CIP was undertaken for comparison with the fungal enzyme and in order to establish a meaningful basis for solution studies of CIP mutants. Proton resonance assignments of haem and haem-linked residues obtained for the cyanide-ligated form of recombinant CIP revealed a high degree of spectral similarity with those of lignin and manganese-dependent peroxidases and extend previously reported NMR data for fungal CIP. The three mutants examined by NMR spectroscopy comprised site-specific substitutions made to a region of the structure believed to form part of the peroxidase haem group access channel for substrate and ligand molecules. Proton resonances of the aromatic side-chains of Phe156 and Phe157 were found to have similar spectral characteristics to those of two phenylalanine residues known to be involved in the binding of aromatic donor molecules to the plant peroxidase, horseradish peroxidase isoenzyme C. The results are discussed in the context of complementary reactivity studies on the mutants in order to develop a more detailed understanding of aromatic donor molecule binding to fungal and plant peroxidases.

G. Weber [(1984) Proc. Natl. Acad. Sci. USA 81, 7098-7102] has inferred that the Monod-Wyman-Changeux (MWC) model for ligand binding by hemoglobin would require (contrary to experimental evidence) that increased ligand binding must promote stabilization of alpha 2 beta 2 tetramers with respect to dissociation into alpha beta dimers. Reexamination of the MWC model, however, in the light of general linkage principles and the specific analysis by G. K. Ackers and M. L. Johnson [(1981) J. Mol. Biol. 147, 559-582] shows that the opposite relation must hold, in agreement with experiment. The T form of the tetramer, with low ligand affinity, must be destabilized and progressively dissociates into the high-affinity dimers, designated D, as ligand binding increases. Each ligand molecule bound shifts the standard Gibbs free energy delta G2T for the D-T equilibrium by approximately 3 kcal/mol in favor of the dimer. Thus, T must exist in (at least) five delta G levels of cooperative free energy as it becomes progressively destabilized by successive binding of ligand molecules. Dissociation of the R tetramer to dimers, in contrast, is independent of the amount of ligand bound, so long as dimers and R-state tetramers possess the same (high) affinity for ligand. While the intrinsic ligand-binding constants of the T and R states (KT and KR) remain unchanged throughout by the postulates of the model, the model should not be regarded as a strictly two-state system in view of the multiple free-energy levels indicated above. The present analysis gives approximate, though not precise, agreement with experimental findings on the dimer-tetramer equilibrium considered by Weber and provides a rationale for interpreting other recent experiments concerning this equilibrium.

The stability of a complex particle (ion or molecule) in solution is determined by the nature of the central atom and the ligands. The most important characteristics of the central atom, determining the stability of the complex compound, are the degree of oxidation (charge on the central ion in the case of ionic complexes), the dimensions, and the electronic structure. In the case of complexes with monatomic ligands, stability is dependent on the same characteristics in the ligand (charge, radius and electronic structure). The strength of binding for ligand molecules and polyatomic ions depends, in addition, on the nature of the atoms directly linked to the central atom, and on the particular features of the structure of the ligand molecule (orion).

DNA stiffening and elongation caused by the binding of ethidium bromide

Ultrasensitive analysis of binding affinity of HIV receptor and neutralizing antibodies using solution-phase electrochemiluminescence assay

Unusual binding of Grb2 protein to a bivalent polyproline-ligand immobilized on a SPR sensor: Intermolecular bivalent binding

Flow cytometry is an essential tool in biology and medicine with applications in immunodeficiency diagnostic, immunosurveillence, organ transplantation in addition to its basic research usage. Before the widespread use of the Fluorescent-Activated Cell Sorting (FACS), the first flow cytometer was invented by Wallace Coulter and was embodied in a remarquably simple device. The Coulter counter principle consists in passing particles in an aperture concurrently with an electric current to measure the related impedance variations. In the fifties, the Coulter counter has established as a gold standard for cell counting and sizing. More recently, thanks to advances in miniaturization, micromachined impedance spectroscopy flow cytometers extended their analysis capabilities to the dielectric properties of cells. This opened the way to label-free and non-invasive methods for cell population differentiations such as leukocytes clustering. However, these flow cytometers were not designed for cell retrieval neither provided the ability to work with a limited number of scarce cells. First, this thesis extends the capability of impedance based flow cytometers to perform single cell isolation on disposable devices. Today, many biological methods are based on single cell isolation. In cell lines development, the gold standard procedure involves serial dilution. However, this approach is time-consuming as it needs to be repeated over several weeks to ensure clonality. In this thesis, a tool enabling single cell isolation in one step and based on impedance spectroscopy is developed. The modeling, designing and testing of a disposable pipette tip integrating a cell sensor based on the Coulter principle is reported. Coupled with an instrumented pipette, this disposable sensing tip enables single cell dispensing. Furthermore, this system allows recording the impedance trace to be used as proof of single cell isolation. Second, this thesis translates the concept of disposable, sterile and low-cost single-cell dispensing device on a standard planar microfabrication technology. Using a planar microfabrication technology enables a better control of the fluidic behavior and permits the integration of more complex features on the dispensing device. However, a disposable device requires a large-scale and cost-effective production method. A planar fabrication method based on the industrially standardized printed circuit board (PCB) manufacturing process is assessed to produce different topologies of flow cytometers with emphasis on the disposable aspect of the devices required for cell culture. Third, this thesis is dedicated to further exploring the cell parameters that can be analyzed by impedance spectroscopy. Ligand-gated ion channels are cellular membrane proteins reacting very specifically and rapidly to the binding of a ligand molecule and modulate the membrane permeability. This cellular mechanism was proposed as the transducing elements for highly sensitive and specific chemical biosensors. However, this implies the engineering challenges of a long-term, automated and integrated cellular electrophysiology monitoring. Finally, in this thesis, the monitoring of ligand-gated ion channel permeability is investigated using impedance spectroscopy as a real-time, non-invasive and label free analytical technique.

Thyroid hormone receptor (THR) belongs to the nuclear receptor (NR) superfamily that is activated by binding of appropriate ligand molecules (thyroid hormones). These receptors directly bind to specific DNA sequences for gene expression, which is essential for metabolism, homeostasis, and the development of organisms, making it an important drug target. Extensive MD-simulation studies of triiodothyronine (T3) docked modeled rnTHRβ1 structures have indicated the presence of twelve conserved water molecules at the DNA-DBD (DNA binding domain) interface. The W1-W5 water centers have been involved in the recognition between the A-chain of DBD to C-chain of DNA, W6 and W7 mediated the interaction between A-chain of DBD and D-chain of DNA, W8 and W9 recognized the B-chain of DBD and C-chain of DNA, and W9-W12 centers conjugated the residues of B-chain of DBD to D-chain of DNA through hydrogen bonds. The conformation flexibility of Phe272 and Met313 residues in the absence of T3 at the LBD (ligand-binding domain) region have been observed and reported.