Determination Of Equilibrium Binding Constants Research Articles

Cupric Ions Selectively Modulate TRAAK-Phosphatidylserine Interactions.

TRAAK and TREK2 are two-pore domain K+ (K2P) channels and are modulated by diverse factors including temperature, membrane stretching, and lipids, such as phosphatidic acid. In addition, copper and zinc, both of which are essential for life, are known to regulate TREK2 and a number of other ion channels. However, the role of ions in the association of lipids with integral membrane proteins is poorly understood. Here, we discover cupric ions selectively modulate the binding of phosphatidylserine (PS) to TRAAK but not TREK2. Other divalent cations (Ca2+, Mg2+, and Zn2+) bind both channels but have no impact on binding PS and other lipids. Additionally, TRAAK binds more avidly to Cu2+ and Zn2+ than TREK2. In the presence of Cu2+, TRAAK binds similarly to PS with different acyl chains, indicating a crucial role of the serine headgroup in coordinating Cu2+. High-resolution native mass spectrometry (MS) enables the determination of equilibrium binding constants for distinct Cu2+-bound stoichiometries and uncovered the highest coupling factor corresponds to a 1:1 PS-to-Cu2+ ratio. Interestingly, the next three highest coupling factors had a ∼1.5:1 PS-to-Cu2+ ratio. Our findings bring forth the role of cupric ions as an essential cofactor in selective TRAAK-PS interactions.

Standard errors or asymmetric confidence intervals of the protein-ligand binding constant

The determination of equilibrium binding constants (Kb) between chemical compounds (ligands) and target proteins in vitro is important for the understanding of biological interaction mechanisms. Accuracy of the data is critical for decision-making in many scientific fields. The Kb is commonly assumed to follow a symmetric normal, Gaussian, distribution, and is often reported as experimental value plus/minus standard deviation. However, in general, the distributions of the measured Kb values (and the equivalent dissociation constant, Kd=1/Kb) and the corresponding Gibbs energy change (ΔGb=-RTlnKb) are all asymmetric to varying degrees.

Binding interactions of hydroxylated polychlorinated biphenyls (OHPCBs) with human hydroxysteroid sulfotransferase hSULT2A1

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants, and exposure to PCBs and their hydroxylated metabolites (OHPCBs) has been associated with various adverse health effects. The mammalian cytosolic sulfotransferases (SULTs) catalyze the sulfation of OHPCBs, and the interaction of OHPCBs with both the SULT1 and SULT2 families of these enzymes has received attention both with respect to metabolic disposition of these molecules and the potential mechanisms for their roles in endocrine disruption. We have previously shown that OHPCBs interact with human hydroxysteroid sulfotransferase hSULT2A1, an enzyme that catalyzes the sulfation of dehydroepiandrosterone (DHEA), other alcohol-containing steroids, bile acids, and many xenobiotics. The objective of our current studies is to investigate the mechanism of inhibition of hSULT2A1 by OHPCBs by combining inhibition kinetics with determination of equilibrium binding constants and molecular modeling of potential interactions. Examination of the effects of fifteen OHPCBs on the sulfation of DHEA catalyzed by hSULT2A1 showed predominantly noncompetitive inhibition patterns. This was observed for OHPCBs that were substrates for sulfation reactions catalyzed by the enzyme as well as those that solely inhibited the sulfation of DHEA. Equilibrium binding experiments and molecular modeling studies indicated that the OHPCBs bind at the binding site for DHEA on the enzyme, and that the observed noncompetitive patterns of inhibition are consistent with binding in more than one orientation to more than one enzyme complex. These results have implications for the roles of SULTs in the toxicology of OHPCBs, while also providing molecular probes of the complexity of substrate/inhibitor interactions with hSULT2A1.

Generally Applicable NMR Titration Methods for the Determination of Equilibrium Constants for Coordination Complexes: Syntheses and Characterizations of Metallacrown Ethers with α,ω-Bis(phosphite)-polyether Ligands and Determination of Equilibrium Binding Constants to Li+

A new metallacrown ether, cis-PdCl2{(2,2′-C12H8O2)P(OCH2CH2)4OP(2,2′-O2H8C12)}, has been synthesized and fully characterized by NMR spectroscopy and X-ray crystallography. A new synthetic method and complete NMR characterization for the previously reported and closely related cis-Mo(CO)4(2,2′-C12H8O2)P(OCH2CH2)4OP(2,2′-O2H8C12) metallacrown ether are also reported. Both complexes are monomeric, exhibiting cis coordination geometries both in solution and in the solid state, and the cis-Mo(CO)4(2,2′-C12H8O2)P(OCH2CH2)4OP(2,2′-O2H8C12) metallacrown ether does not appear to undergo cis–trans isomerization in the presence of HgCl2. These results are surprising because the closely related PdCl2(Ph2P(CH2CH2O)nCH2CH2PPh2) (n = 3–5) metallacrown ethers exhibit both cis–trans and monomer–cyclic oligomer equilibria in solution, and cis-Mo(CO)4 (Ph2P(CH2CH2O)nCH2CH2PPh2) (n = 3–5) metallacrown ethers rapidly undergo cis–trans isomerization in the presence of HgCl2. The coordination of Li+ cations to the cis-Mo(CO)4(2...

Studying the Salt Dependence of the Binding of σ70 and σ32 to Core RNA Polymerase Using Luminescence Resonance Energy Transfer

The study of protein-protein interactions is becoming increasingly important for understanding the regulation of many cellular processes. The ability to quantify the strength with which two binding partners interact is desirable but the accurate determination of equilibrium binding constants is a difficult process. The use of Luminescence Resonance Energy Transfer (LRET) provides a homogeneous binding assay that can be used for the detection of protein-protein interactions. Previously, we developed an LRET assay to screen for small molecule inhibitors of the interaction of σ70 with theβ' coiled-coil fragment (amino acids 100–309). Here we describe an LRET binding assay used to monitor the interaction of E. coli σ70 and σ32 with core RNA polymerase along with the controls to verify the system. This approach generates fluorescently labeled proteins through the random labeling of lysine residues which enables the use of the LRET assay for proteins for which the creation of single cysteine mutants is not feasible. With the LRET binding assay, we are able to show that the interaction of σ70 with core RNAP is much more sensitive to NaCl than to potassium glutamate (KGlu), whereas the σ32 interaction with core RNAP is insensitive to both salts even at concentrations >500 mM. We also find that the interaction of σ32 with core RNAP is stronger than σ70 with core RNAP, under all conditions tested. This work establishes a consistent set of conditions for the comparison of the binding affinities of the E.coli sigma factors with core RNA polymerase. The examination of the importance of salt conditions in the binding of these proteins could have implications in both in vitro assay conditions and in vivo function.

Measuring Protein Interactions by Optical Biosensors

In recent years, optical evanescent wave biosensors have been used to characterize protein-protein interactions, including determination of equilibrium binding constants and bimolecular rate constants. This surface binding technique can provide information about chemical on-rate constants, the lifetimes of complexes formed, and the time course of the signal. This unit provides a thorough, well-illustrated discussion of the principles of optical biosensors, experimental design, ligand immobilization, experimental data analysis, and common obstacles and possible solutions.

Compensation for Loss of Ligand Activity in Surface Plasmon Resonance Experiments

The determination of equilibrium binding constants is an important aspect of the analysis of protein–protein interactions. In recent years surface plasmon resonance experiments (e.g., with a BIAcore instrument) have provided a valuable experimental approach to determining such constants. The standard method is based on measuring amounts of analyte bound at equilibrium for different analyte concentrations. During the course of a typical surface plasmon resonance experiment the measured equilibrium levels for a given analyte concentration often decrease. This appears to be due to a loss of activity of the protein coupled to the sensor chip or other phenomena. The loss in signal can lead to an erroneous determination of the equilibrium constant. A data analysis approach is introduced that aims to compensate for the loss of activity so that its influence on the results of the experiments is reduced.

Scintillating microtitration plates, a platform for hormone and DNA binding assays. Model experiments with the human estrogen receptor

The estrogen receptor is an important drug target and an allosteric binder that binds hormones, DNA and proteins at different sites on its surface. Recombinantly produced human estrogen receptor (hER) and estrogen receptor hormone binding domain (hER-HBD) was used in conjunction with a novel platform for ligand binding studies, scintillating microtitration plates, to obtain quantitative binding data. Radioligand binding was determined in a multi-detector scintillation counter designed for the microtitration plate format. Methodology was developed that allows determination of equilibrium binding constants and of kinetic rate constants for both hormone-and DNA-binding. The results obtained show that the methodology is valid with regard to binding characteristics of the estrogen receptor. The assay format with scintillating microtitration plates has a major advantage over regular liquid scintillation counting or other solid-phase scintillant procedures as the vial itself is the means for sample holding and detection of radioactivity. Thus, the scintillating microtitration plates are especially suited for high capacity equilibrium and kinetic binding assays.

Structural requirements of tetracycline-Tet repressor interaction: determination of equilibrium binding constants for tetracycline analogs with the Tet repressor

We used the Tn10-encoded Tet repressor, which has a highly specific binding capacity for tetracycline, to probe contacts between the drug and protein by chemical interference studies of the antibiotic. For that purpose, the equilibrium association constants of modified tetracyclines with the Tet repressor and Mg2+ cations were determined quantitatively. The results confirm the previous notion that Mg2+ probably binds with the oxygens at positions 11 and 12 and is absolutely required for protein-drug recognition. Modifications were introduced at positions seven, six, five, and four of the drug, and anhydrotetracycline was also studied. Substitutions or eliminations of functions at these positions influenced binding to the Tet repressor up to 35-fold. The introduction of an azido function at position seven in 7-azidotetracycline and epimerization of the substituents at position four in 4-epitetracycline lead to a 2- or 25-fold reduction, respectively, of Tet repressor affinity in those compounds. Anhydrotetracycline bound about 35-fold more strongly than tetracycline did, indicating that the oxygen at position 11 may be involved in Tet repressor recognition. This increased binding is in contrast to the lower antibiotic activity of anhydrotetracycline and indicates that the Tet repressor and ribosomes recognize the drug differently.

The rapid determination of binding constants for antiviral antibodies by a radioimmunoassay. An analysis of the interaction between hybridoma proteins and influenza virus

A method is described that allows the rapid determination of equilibrium binding constants for antibody-antigen reactions. The method, based upon the radioimmunoassay procedure of Rosenthal et al. (1973), has been applied to the interaction of monoclonal antiviral hybridoma antibodies with a panel of influenza viruses. Equilibrium binding constants with values from 10 6 M −1 to 10 10 M −1 have been observed with errors of approximately ±6.5%. An independent determination by equilibrium dialysis of the binding constant of one hybridoma antibody is in excellent agreement with the results presented here. Heterogeneity constants for the reaction pairs are also presented and are consistent with the interaction of monoclonal antibodies with homogeneous protein determinants.

The interaction of 7-substituted actinomycin D analogs with DNA

The interaction of actinomycin D and three new 7-substituted analogs with calf thymus DNA has been studied by a number of physical techniques. The methods utilized in this investigation include visible absorption spectrometry and ultrafiltration methodology for the determination of equilibrium binding constants; viscometry; and circular dichroism. The studies show that the 7-substituted actinomycin D analogs retain the G · C base pair specific DNA binding demonstrated by actinomycin D. The mode of binding to native DNA, despite substitution at position 7, is practically unaltered. The retention of this binding specificity by these analogs seems to be unaffected by changes in the electron properties of the chromophore.

A comparison of the interactions of galactosyltransferase with a glycoprotein substrate (Ovalbumin) and with alpha-lactalbumin.

Sedimentation velocity ultracentrifugation and other procedures have been used to investigate macromolecular interactions of bovine colostrum galactosyltransferase with a glycoprotein substrate (ovalbumin) and with the lactose synthase regulatory protein, alpha-lactalbumin. The determination of equilibrium binding constants for these interactions and the effects of ligands and combinations of ligands on the equilibria have clarified several aspects of the mechanism of galactosyltransferase and its regulation by alpha-lactalbumin. 1. The attachment of Mn2+ at the tight binding site on galactosyltransferase (site I, Kd 2.3 muM) is an essential prerequisite for interactions with ovalbumin and with alpha-lactalbumin. 2. The attachment of Mn2+ or Ca2+ at the weaker metal binding site (site II, Kd 1 to 2 mM) does not significantly affect the interaction of galactosyltransferase with either protein. This is consistent with the hypothesis derived from kinetic studies that site II is functionally connected with the binding of UDP-derivatives. 3. While the binding of ovalbumin to galactosyltransferase in the presence of Mn2+ alone can be observed by ultracentrifugation, this interaction is too weak to cause binding of galactosyltransferase to ovalbumin-Sepharose. Binding to ovalbumin-Sepharose could only be detected by affinity chromatography in the presence of both Mn2+ (10 mM) and UDP (0.3 MM). Sedimentation studies showed that the association of galactosyltransferase with ovalbumin is pressure-dependent, and that the presence of UDP in the complex increases the equilibrium association constant by a factor of 46. The enzyme Mn2+-UDP-ovalbumin complex has unusual hydrodynamic properties. 4. The presence of saturating concentrations of UDP-galactose potentiates the binding of alpha-lactalbumin at high concentrations of Mn2+, as shown by a 25-fold increase in the association constant. Competitive inhibition by alpha-lactalbumin, with respect to ovalbumin that is observed by steady state kinetics, is attributed to the mutally exclusive binding of the proteins with an enzyme complex containing Mn2+ and UDP-galactose. 5. Monsaccharides (N-acetylglucosamine and glucose) strongly enhance the binding of alpha-lactalbumin to enzyme complexes containing Mn2+, in the presence or absence of UDP-glucose. The binding of alpha-lactalbumin and monosaccharide to form enzyme complexes containing both is random, and evaluation of the four associated equilibrium constants shows that the binding is strongly synergistic. 6. Although the significance of some of the many equilibria studied cannot be ascertained, it appears than an element of randomness may be present in reactions catalyzed by galactosyltransferase...