A combinatorial biophysical approach; FTSA and SPR for identifying small molecule ligands and PAINs.

Biophysical methods have emerged as attractive screening techniques in drug discovery both as primary hit finding methodologies, as in the case of weakly active compounds such as fragments, and as orthogonal methods for hit validation for compounds discovered through conventional biochemical or cellular assays. Here we describe a dual method employing fluorescent thermal shift assay (FTSA), also known as differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR), to interrogate ligands of the kinase p38α as well as several known pan-assay interference compounds (PAINs) such as aggregators, redox cyclers, and fluorescence quenchers. This combinatorial approach allows for independent verification of several biophysical parameters such as KD, kon, koff, ΔG, ΔS, and ΔH, which may further guide chemical development of a ligand series. Affinity values obtained from FTSA curves allow for insight into compound binding compared with reporting shifts in melting temperature. Ligand-p38 interaction data were in good agreement with previous literature. Aggregators and fluorescence quenchers appeared to reduce fluorescence signal in the FTSAs, causing artificially high shifts in Tm values, whereas redox compounds caused either shifts in affinity that did not agree between FTSA and SPR or a depression of FTSA signal.

Thermodynamics of DNA binding and condensation: isothermal titration calorimetry and electrostatic mechanism.

The thermodynamics of binding of the trivalent cations cobalt hexammine and spermidine to plasmid DNA was studied by isothermal titration calorimetry. Two stages were observed in the course of titration, the first attributed to cation binding and the second to DNA condensation. A standard calorimetric data analysis was extended by applying an electrostatic binding model, which accounted for most of the observed data. Both the binding and condensation reactions were entropically driven (TDeltaS approximately +10 kcal/mol cation) and enthalpically opposed (DeltaH approximately +1 kcal/mol cation). As predicted from their relative sizes, the binding constants of the cations were indistinguishable, but cobalt hexammine had a much greater DNA condensing capacity because it is more compact than spermidine. The dependence of both the free energy of cobalt hexammine binding and the critical cobalt hexammine concentration for DNA condensation on temperature and monovalent cation concentration followed the electrostatic model quite precisely. The heat capacity changes of both stages were positive, perhaps reflecting both the temperature dependence of the dielectric constant of water and the burial of polar surfaces. DNA condensation occurred when about 67 % of the DNA phosphate charge was neutralized by cobalt hexammine and 87 % by spermidine. During condensation, the remaining DNA charge was neutralized.

Nickel resistance in Escherichia coli V38 is dependent on the concentration used for induction.

Strain Escherichia coli V38 resistant to 4 mM NiCl2 was isolated from the city sewage sludge. It showed low nickel accumulation by cells and nickel ion efflux. Cells were pregrown (induced) overnight in the presence of Ni2+, then the culture was kept on ice for 20-30 min and transferred to 37 degrees C for further incubation. When the Ni2+ concentration during growth was the same as during incubation, there was no noticeable accumulation of Ni2+. When the Ni2+ concentration during incubation was higher than that used for induction, uptake of 63Ni2+ and delayed efflux were seen. The uptake and delay of both efflux and growth were directly proportional to the difference between the concentrations used for induction and incubation. Active nickel ion uptake was seen in cells taken from cultures in the delayed efflux period.

Thermodynamics of the hydrophobic effect. III. Condensation and aggregation of alkanes, alcohols, and alkylamines.

Knowledge of the energetics of the low solubility of non-polar compounds in water is critical for the understanding of such phenomena as protein folding and biomembrane formation. Solubility in water can be considered as one leg of the three-part thermodynamic cycle - vaporization from the pure liquid, hydration of the vapor in aqueous solution, and aggregation of the substance back into initial pure form as an immiscible phase. Previous studies on the model compounds n-alkanes, 1-alcohols, and 1-aminoalkanes have noted that the thermodynamic parameters (Gibbs free energy, DeltaG; enthalpy, DeltaH; entropy, DeltaS; and heat capacity, DeltaC(p)) associated with these three processes are generally linear functions of the number of carbons in the alkyl chains. Here we assess the accuracy and limitations of the assumption of additivity of CH(2) group contributions to the thermodynamic parameters for vaporization, hydration, and aggregation. Processes of condensation from pure gas to liquid and aqueous solution to aggregate are compared. Hydroxy, amino, and methyl headgroup contributions are estimated, liquid and solid aggregates are distinguished. Most data in the literature were obtained for compounds with short aliphatic hydrocarbon tails. Here we emphasize long aliphatic chain behavior and include our recent experimental data on long chain alkylamine aggregation in aqueous solution obtained by titration calorimetry and van't Hoff analysis. Contrary to what is observed for short compounds, long aliphatic compound aggregation has a large exothermic enthalpy and negative entropy.

Thermodynamics of the hydrophobic effect. I. Coupling of aggregation and pK(a) shifts in solutions of aliphatic amines.

Long aliphatic hydrocarbon chains aggregate in aqueous solution due to the hydrophobic effect, forming structures such as micelles and membranes, while amino groups titrate at basic pH. These two biologically important behaviors are linked in alkylamines, in which the pK(a) of the amino group is shifted downward by aggregation. In this paper we study the thermodynamics of these coupled processes, following aggregation by observing alkylamine pH titration behavior. The magnitude of the shift depended on the aliphatic chain length and on the concentration of alkylamine: longer chains and higher concentrations lowered the pK(a) to a greater extent. Gibbs free energies of protonation and aggregation were calculated from the pK(a) shifts. Enthalpies, entropies, and heat capacities were estimated by van't Hoff analysis from the pK(a) shift dependencies on temperature. However, the results were less precise than the calorimetrically measured values, as described in the following article. A model to calculate titration curves, pK(a) shifts, and aggregation of uncharged alkylamines as a function of aliphatic chain length, concentration, and temperature is presented.

Thermodynamics of the hydrophobic effect. II. Calorimetric measurement of enthalpy, entropy, and heat capacity of aggregation of alkylamines and long aliphatic chains.

The thermodynamics of long aliphatic chain alkylamine aggregation in aqueous solution was studied by isothermal titration calorimetry (ITC). Protonated alkylammonium cations with linear aliphatic chains of 10-14 carbon atoms were fully soluble in aqueous solution at the beginning of titration, but practically insoluble after deprotonation by titrating with sodium hydroxide. The alkylamines aggregated and precipitated during the reaction, enabling direct measurement of the enthalpy of aggregation. The enthalpy of aggregation became increasingly exothermic upon increasing the chain length. Hydrophobic aggregation was enthalpy-driven and entropy-opposed for alkylamines with 12-14 carbon atoms at room temperature. Direct observation of hydrophobic aggregation by ITC at constant temperature and pressure provided more accurate thermodynamic parameters than obtainable from van't Hoff analysis. Aggregation into liquid or solid phases could be distinguished by ITC, but not by van't Hoff analysis of alkylamine solubility data.

1-Anilino-8-naphthalene sulfonate anion-protein binding depends primarily on ion pair formation.

The ANS- (1-anilino-8-naphthalene sulfonate) anion is strongly, dominantly bound to cationic groups of water-soluble proteins and polyamino acids through ion pair formation. This mode of ANS- binding, broad and pH dependent, is expressed by the quite rigorous stoichiometry of ANS- bound with respect to the available summed number of H+ titrated lysine, histidine, and arginine groups. By titration calorimetry, the integral or overall enthalpies of ANS- binding to four proteins, bovine serum albumin, lysozyme, papain, and protease omega, were arithmetic sums of individual ANS(-)-polyamino acid sidechain binding enthalpies (polyhistidine, polyarginine, polylysine), weighted by numbers of such cationic groups of each protein (additivity of binding enthalpies). ANS- binding energetics to both classes of macromolecules, cationic proteins and synthetic cationic polyamino acids, is reinforced by the organic moiety (anilinonaphthalene) of ANS-. In a much narrower range of binding, where ANS- is sometimes assumed to act as a hydrophobic probe, ANS- may become fluorescent. However, the broad overall range is sharply dependent on electrostatic, ion pair formation, where the organic sulfonate group is the major determinant of binding.

Selective precipitation of proteins.

Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset of proteins from a protein solution, or as a very specific method to recover a single protein of interest from a purification step. This unit describes a number of methods suitable for selective precipitation. In each of the protocols that are outlined, the physical or chemical basis of the precipitation process, the parameters that can be varied for optimization, and the basic steps for developing an optimized precipitation are described.

Lectin coprecipitative isolation from crudes by Little Rock Orange ligand.

Matrix ligands are intended for upstream use with dilute crudes on a large scale, splitting out sought-for proteins by coprecipitating them as dense, protected aggregates. Matrix ligand coprecipitation is rapidly, quantitatively reversible, by pH shifting and trapping matrix ligands on ion exchange resin, releasing the sought-for protein. Four lectins, wheat germ agglutinin, peanut lectin, concanavalin A, and Phaseolus vulgaris (red kidney bean) lectins, were coprecipitated from crude extracts, 0.05 to 0.4% crude protein, in a single step using Little Rock Orange matrix ligand. All were compared in specific activities (erythrocyte agglutination) and in SDS-PAGE analysis with the four corresponding commercial lectins purified by affinity chromatography. All four matrix-coprecipitated ligands were specifically active within range of the corresponding vendor (Sigma Co.) affinity chromatography-purified lectins. The matrix ligand coprecipitative technique requires optimization of ligand-protein (crude) ratios, denoted y, and determination of suitable pH ranges for coprecipitation relative to lectin isoelectric pH. These parameters control electrostatic ion pair association: ligand head anion binding to cationic target proteins. The coprecipitative and protective powers of new ligands like Little Rock Orange, their ability to scavenge sought-for lectins from dilute crudes, depend on ligand organic tail-tail association. After the strong anion heads of ligands bind to cationic proteins, their organic tails stack and draw the ligand-protein complexes together as aggregated coprecipitates.

Coprecipitation of proteins with matrix ligands: scaleable protein isolation.

Matrix ligands are agents for isolating proteins out of dilute crudes by coprecipitating proteins. The ligands have a strong anion sulfonate head which initiates binding to proteins having a positive net charge, ZH+ approximately 5-20. Initial binding tightens protein conformation and starts to squeeze water from conformationally motile proteins. The tails are stackable hydrophobic organic groups, azoaromatic dyes which draw protein-ligand complexes together. Proteins coprecipitate as guests, in the ligand host matrix. In addition to stacking, ligand tails displace water because of their bulk, and lower the average dielectric constant near charged groups, which reinforces the electrostatic component of binding. Matrix ligands protect proteins, scavenge them from dilute crudes (0.01-0.1 per cent protein), and densify coprecipitates. Detergent ions in low concentrations, 10(-4)-10(-5) M also sometimes serve as coprecipitating agents, entangling their tails but probably not stacking. Divalent metal ions, Zn++, sometimes are useful auxiliary agents. Preparative scaleability from crudes is demonstrated starting from 100-200 g of raw peanuts and raw pineapple to coprecipitate a lectin and bromelain enzyme respectively in 1-2 h with 80-90 per cent activity yields. Ligands are released from coprecipitates by shifting the pH and trapping the ligands with exchange resins. Protein conformation tightening in solution is seen by viscosity measurements.

1-anilino-8-naphthalene sulfonate as a protein conformational tightening agent.

1-Anilino-8-naphthalene sulfonate (ANS) anion is conventionally considered to bind to preexisting hydrophobic (nonpolar) surfaces of proteins, primarily through its nonpolar anilino-naphthalene group. Such binding is followed by an increase in ANS fluorescence intensity, similar to that occurring when ANS is dissolved in organic solvents. It is generally assumed that neither the negative sulfonate charge on the ANS, nor charges on the protein, participate significantly in ANS-protein interaction. However, titration calorimetry has demonstrated that most ANS binding to a number of proteins occurs through electrostatic forces, in which ion pairs are formed between ANS sulfonate groups and cationic groups on the proteins (D. Matulis and R. E. Lovrien, Biophys. J., 1998, Vol. 74, pp. 1-8). Here we show by viscometry and diffusion coefficient measurements that bovine serum albumin and gamma-globulin, starting from their acid-expanded, most hydrated conformations, undergo extensive molecular compaction upon ANS binding. As the cationic protein binds negatively charged ANS anion it also takes up positively charged protons from water to compensate the effect of the negative charge, and leaves the free hydroxide anions in solution thus shifting pH upward (the Scatchard-Black effect). These results indicate that ANS is not always a definitive reporter of protein molecular conformation that existed before ANS binding. Instead, ANS reports on a conformationally tightened state produced by the interplay of ionic and hydrophobic characters of both protein and ligand.