Understanding the physical basis of the salt dependence of the electrostatic binding free energy of mutated charged ligand-nucleic acid complexes

The predictions of the derivative of the electrostatic binding free energy of a biomolecular complex, ΔG_el, with respect to the logarithm of the 1:1 salt concentration, d(ΔG_el)/d(ln[NaCl]), SK, by the Poisson-Boltzmann equation, PBE, are very similar to those of the simpler Debye-Hückel equation, DHE, because the terms in the PBE's predictions of SK that depend on the details of the dielectric interface are small compared to the contributions from long-range electrostatic interactions. These facts allow one to obtain predictions of SK using a simplified charge model along with the DHE that are highly correlated with both the PBE and experimental binding data. The DHE-based model developed here, which was derived from the generalized Born model, explains the lack of correlation between SK and ΔG_el in the presence of a dielectric discontinuity, which conflicts with the popular use of this supposed correlation to parse experimental binding free energies into electrostatic and nonelectrostatic components. Moreover, the DHE model also provides a clear justification for the correlations between SK and various empirical quantities, like the number of ion pairs, the ligand charge on the interface, the Coulomb binding free energy, and the product of the charges on the complex's components, but these correlations are weak, questioning their usefulness.     

Structural investigation into the inhibitory mechanisms of indomethacin and its analogues towards human glyoxalase I

In the present work, a combined study of kinetic analysis, molecular docking, and molecular dynamics simulations on indomethacin and its analogues is performed to better understand their inhibitory mechanisms towards human glyoxalase I (GLOI). A remarkable correlation (R² = 0.974) was observed for six inhibitors including indomethacin between their experimental inhibitory affinities and predicted binding free energy parameter (ΔG_bind,pred). This suggests that ΔG_bind,pred of a GLOI/inhibitor complex can be efficiently used to interpolate the experimental inhibitory affinity of a ligand of similar nature in the GLOI enzyme system. Energetic analyses revealed that electrostatic contribution plays an important role in their inhibitory mechanisms, which reflects the significant contribution of the coordination bond between zinc and ligands. The present work highlights that indomethacin is a promising lead as GLOI inhibitors for further development since it may bind all subsites in the active site pocket of GLOI and stabilize the flexible loop (152-159).     

Binding thermodynamics of phosphorylated inhibitors to triosephosphate isomerase and the contribution of electrostatic interactions

Electrostatic interactions have a central role in some biological processes, such as recognition of charged ligands by proteins. We characterized the binding energetics of yeast triosephosphate isomerase (TIM) with phosphorylated inhibitors 2-phosphoglycollate (2PG) and phosphoglycolohydroxamate (PGH). We determined the thermodynamic parameters of the binding process (K_b, ΔG_b, ΔH_b, ΔS_b and ΔC_p) with different concentrations of NaCl, using fluorimetric and calorimetric titrations in the conventional mode of ITC and a novel method, multithermal titration calorimetry (MTC), which enabled us to measure ΔC_p in a single experiment. We ruled out specific interactions of Na⁺ and Cl^- with the native enzyme and did not detect significant linked protonation effects upon the binding of inhibitors. Increasing ionic strength (I) caused K_b, ΔG_b and ΔH_b to become less favorable, while ΔS_b became less unfavorable. From the variation of K_b with I, we determined the electrostatic contribution of TIM−2PG and TIM−PGH to ΔG_b at I = 0.06 M and 25 °C to be 36% and 26%, respectively. The greater affinity of PGH for TIM is due to a more favorable ΔH_b compared to 2PG (by 19-24 kJ mol^-1 at 25 °C). This difference is compatible with PGH establishing up to five more hydrogen bonds with TIM. Both binding ΔC_ps were negative, and less negative with increasing ionic strength. ΔC_ps at I = 0.06 M were much more negative than predicted by surface area models. Water molecules trapped in the interface when ligands bind to protein could explain the highly negative ΔCps. Thermodynamic binding functions for TIM−2PG changed more with ionic strength than those for TIM−PGH. This greater dependence is consistent with linked, but compensated, protonation equilibriums yielding the dianionic species of 2PG that binds to TIM, process that is not required for PGH.     

Molecular dynamics simulation study of binding affinity of thieno[2,3-<Emphasis Type="Italic">b</Emphasis>]benzo[1,8]naphthyridine derivatives to DNA

DNA binding position and binding affinity of drugs are important information that helps medicinal chemists in synthesis of new drugs. We used molecular docking and molecular dynamics simulation to reveal binding strength of thieno[2,3-b]benzo[1,8]naphthyridine derivatives to DNA. Molecular docking showed that molecules with more steric hindrance select groove position in DNA structure. Other molecules are intercalated between base pairs of GC and AT. Restrained electrostatic potential (RESP) charges, root mean square deviation (RMSD), and total potential analyses were performed. RMSD and total potential analyses showed that all simulations have stability for MMGBSA analysis. Binding affinity of all drugs was derived via MMGBSA analysis. Thermodynamics analysis showed that binding affinity of groove binding drugs is less than that of intercalating ones. Also, it was found that a linear relationship exists between RESP charges and ΔG _pred. Additionally, our results demonstrated the highest affinity for molecules carrying substituent groups of–OCH₃ and–CH₃.     

Formation of a wrapped DNA-protein interface: experimental characterization and analysis of the large contributions of ions and water to the thermodynamics of binding IHF to H' DNA

To characterize driving forces and driven processes in formation of a large-interface, wrapped protein-DNA complex analogous to the nucleosome, we have investigated the thermodynamics of binding the 34-base pair (bp) H′ DNA sequence to the Escherichia coli DNA-remodeling protein integration host factor (IHF). Isothermal titration calorimetry and fluorescence resonance energy transfer are applied to determine effects of salt concentration [KCl, KF, K glutamate (KGlu)] and of the excluded solute glycine betaine (GB) on the binding thermodynamics at 20 °C. Both the binding constant K_obs and enthalpy ΔH°_obs depend strongly on [salt] and anion identity. Formation of the wrapped complex is enthalpy driven, especially at low [salt] (e.g., ΔH^o_obs = − 20.2 kcal·mol^− 1 in 0.04 M KCl). ΔH°_obs increases linearly with [salt] with a slope (dΔH°_obs/d[salt]), which is much larger in KCl (38 ± 3 kcal·mol^− 1 M^− 1) than in KF or KGlu (11 ± 2 kcal·mol^− 1 M^− 1). At 0.33 M [salt], K_obs is approximately 30-fold larger in KGlu or KF than in KCl, and the [salt] derivative SK_obs = dlnK_obs/dln[salt] is almost twice as large in magnitude in KCl (− 8.8 ± 0.7) as in KF or KGlu (− 4.7 ± 0.6).A novel analysis of the large effects of anion identity on K_obs, SK_obs and on ΔH°_obs dissects coulombic, Hofmeister, and osmotic contributions to these quantities. This analysis attributes anion-specific differences in K_obs, SK_obs, and ΔH°_obs to (i) displacement of a large number of water molecules of hydration [estimated to be 1.0(± 0.2) × 10³] from the 5340 Å² of IHF and H′ DNA surface buried in complex formation, and (ii) significant local exclusion of F⁻ and Glu⁻ from this hydration water, relative to the situation with Cl⁻, which we propose is randomly distributed. To quantify net water release from anionic surface (22% of the surface buried in complexation, mostly from DNA phosphates), we determined the stabilizing effect of GB on K_obs: dlnK_obs/d[GB]  = 2.7 ± 0.4 at constant KCl activity, indicating the net release of ca. 150 H₂O molecules from anionic surface.     

Hydrophobic interactions of DNA with long-chain amines.

The binding ratio, Γ_a, for several long-chain amines to calf-thymus DNA was measured as function of the ligand concentration, C, using the equilibrium dialysis method. The different amines used in the binding experiments at constant temperature were dodecyl trimethyl ammonium bromide (DTAB), myristyl trimethyl ammonium bromide (MTAB), cetyl trimethyl ammonium bromide (CTAB), and cetyl pyridinium chloride (CPCL). The formation and dissociation of the saturated DNA–amine complex were reversible. The initial slope of the binding isotherm decreased sharply with the reduction of the electrostatic effect as a result of the increase of the ionic strength of the medium. A sharp inflexion region was noted in the binding isotherm where the ligands bound in significant numbers may undergo hydrophobic interactions with each other. Γ_a increased with C until a maximum value, Γ_a^m, was reached, beyond which binding slowly decreased with an increase of concentration. Both Γ_a^m and Γ_a increased significantly with the increase of the hydrocarbon chain length of a ligand. The free energy change ΔG^m for each saturated DNA–amine complex was evaluated on the basis of a thermodynamic relation and the standard state for binding was defined. The average free energy change for the binding per CH₂ group of the amine was found to be ?1550 cal/mol. The difference between ΔG^m for CTAB and CPCL was examined on the basis of the structural difference of their head groups. The binding isotherms for MTAB and CPCL were obtained from the binding data at 15, 30, and 45°C. The binding increased with increasing temperature. From the plot of ΔG^m/T vs 1/T, the changes in enthalpy and entropy due to the binding were evaluated for MTAB and CPCL. The binding reactions in these two cases were driven primarily by the entropy change due to the hydrophobic interaction. Standard free energy changes ΔG₀^m for the unsaturated complexes were close to ΔG^m for the saturated complexes. The binding isotherms also depended on the nature of the neutral salt of the medium. At a given salt concentration, the order of the binding of the inorganic salts was as follows: KCl > NaCl > LiCl > Na₂SO₄ > MgCl₂. The effect of pH on binding was also examined. The importance of these results on the formation of the reconstituted and natural nucleohistone complexes is discussed.     

Quantitative estimates of steric effects: Intramolecular strain energy effects on the activation energy for aquation of some trans-dichlorotetraaminecobalt(III) complexes

In an effort to quantitatively estimate steric contributions to the aquation rates of a series of structurally related cobalt(III) tetraamine complexes, strain energy minimization calculations have been performed on the reactant and some plausible transition state structures. Free energies of activation ΔG*_obs, are factored as: ΔG*_obs, = ΔG*_bb + ΔG*_strain + Δ_G*_CF + ΔG*_solvation + … where ΔG*_bb is the free energy change associated with bond breaking, ΔG*_solvation is the solvation free energy difference between the reactant and a proposed transition stare, ΔG*_CF is the difference in crystal field stabilization between the reactant and a proposed transition state, and ΔG*_strain is the strain energy difference between the reactant complex and a proposed transition state. The activation energy for the aquation of a hypothetical ‘strain free’ complex is defined as ΔG*_int and reflects the energy required for the bond breaking step with all other terms. For the cations trans-(RR,SS)-dichloro-1,8- diamino-3,6-diazaoctanecobalt(III)(trans [Co(2,2,2- tet)Cl₂]⁺), trans-(RR,SS)- or trans-(RS)-dichloro-1.9- diamino-3,7-diazanonanecobalt(III)(trans [Co(2,3,2- tet)Cl₂]⁺ and trans-(RS)-dichloro-1,10-diamino-4,7- diazadecanecobalt(III)(trans[Co(3,2,3-tet)Cl₂]⁺) ΔG*_int is found to be a constant 123 kJ/mol. For the trans-dichlorocobalt(III) complexes with the ligands 1,4,7,10-tetraazacyclotridecane([13]-ane-N₄), 1,4,8, 11-tetraazacyclotetradecane([14]-ane-N₄), 1,4,8,12- tetraazacyclopentadecane([15]-ane-N₄) and 1,5,9,13- tetraazacyclohexadecane([16]-ane-N₄), ΔG*_int lies in the range 133–139 kJ/mol.     

A Mechanism of Modulating the Direction of Flagellar Rotation in Bacteria by Fumarate and Fumarate Reductase

Fumarate, an electron acceptor in anaerobic respiration of Escherichia coli, has an additional function of assisting the flagellar motor to shift from counterclockwise to clockwise rotation, with a consequent modulation of the bacterial swimming behavior. Fumarate transmits its effect to the motor via the fumarate reductase complex (FrdABCD), shown to bind to FliG—one of the motor’s switch proteins. How binding of the FrdABCD respiratory enzyme to FliG enhances clockwise rotation and how fumarate is involved in this activity have remained puzzling. Here we show that the FrdA subunit in the presence of fumarate is sufficient for binding to FliG and for clockwise enhancement. We further demonstrate by in vitro binding assays and super-resolution microscopy in vivo that the mechanism by which fumarate-occupied FrdA enhances clockwise rotation involves its preferential binding to the clockwise state of FliG (FliG_cw). Continuum electrostatics combined with docking analysis and conformational sampling endorsed the experimental conclusions and suggested that the FrdA–FliG_cw interaction is driven by the positive electrostatic potential generated by FrdA and the negatively charged areas of FliG. They further demonstrated that fumarate changes FrdA’s conformation to one that can bind to FliG_cw. These findings also show that the reason for the failure of the succinate dehydrogenase flavoprotein SdhA (an almost-identical analog of FrdA shown to bind to FliG equally well) to enhance clockwise rotation is that it has no binding preference for FliG_cw. We suggest that this mechanism is physiologically important as it can modulate the magnitude of ΔG⁰ between the clockwise and counterclockwise states of the motor to tune the motor to the growth conditions of the bacteria.     

A Role for Weak Electrostatic Interactions in Peripheral Membrane Protein Binding

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is a secreted virulence factor that binds specifically to phosphatidylcholine (PC) bilayers containing negatively charged phospholipids. BtPI-PLC carries a negative net charge and its interfacial binding site has no obvious cluster of basic residues. Continuum electrostatic calculations show that, as expected, nonspecific electrostatic interactions between BtPI-PLC and membranes vary as a function of the fraction of anionic lipids present in the bilayers. Yet they are strikingly weak, with a calculated ΔG_el below 1 kcal/mol, largely due to a single lysine (K44). When K44 is mutated to alanine, the equilibrium dissociation constant for small unilamellar vesicles increases more than 50 times (∼2.4 kcal/mol), suggesting that interactions between K44 and lipids are not merely electrostatic. Comparisons of molecular-dynamics simulations performed using different lipid compositions reveal that the bilayer composition does not affect either hydrogen bonds or hydrophobic contacts between the protein interfacial binding site and bilayers. However, the occupancies of cation-π interactions between PC choline headgroups and protein tyrosines vary as a function of PC content. The overall contribution of basic residues to binding affinity is also context dependent and cannot be approximated by a rule-of-thumb value because these residues can contribute to both nonspecific electrostatic and short-range protein-lipid interactions. Additionally, statistics on the distribution of basic amino acids in a data set of membrane-binding domains reveal that weak electrostatics, as observed for BtPI-PLC, might be a less unusual mechanism for peripheral membrane binding than is generally thought.     

A hill-type muscle model expansion accounting for effects of varying transverse muscle load

Recent studies demonstrated that uniaxial transverse loading (F_G) of a rat gastrocnemius medialis muscle resulted in a considerable reduction of maximum isometric muscle force (ΔF_im). A hill-type muscle model assuming an identical gearing G between both ΔF_im and F_G as well as lifting height of the load (Δh) and longitudinal muscle shortening (Δl_CC) reproduced experimental data for a single load.Here we tested if this model is able to reproduce experimental changes in ΔF_im and Δh for increasing transverse loads (0.64 N, 1.13 N, 1.62 N, 2.11 N, 2.60 N). Three different gearing ratios were tested: (I) constant G_c representing the idea of a muscle specific gearing parameter (e.g. predefined by the muscle geometry), (II) G_exp determined in experiments with varying transverse load, and (III) G_f that reproduced experimental ΔF_im for each transverse load.Simulations using G_c overestimated ΔF_im (up to 59%) and Δh (up to 136%) for increasing load. Although the model assumption (equal G for forces and length changes) held for the three lower loads using G_exp and G_f, simulations resulted in underestimation of ΔF_im by 38% and overestimation of Δh by 58% for the largest load, respectively. To simultaneously reproduce experimental ΔF_im and Δh for the two larger loads, it was necessary to reduce F_im by 1.9% and 4.6%, respectively. The model seems applicable to account for effects of muscle deformation within a range of transverse loading when using a linear load-dependent function for G.     

Effects of salts on the nonequivalent stability of the α-helices of isomeric block copolypeptides

The stability of the α-helices of isomeric block copolypeptides is nonequivalent, as reported previously. In order to explore the origin of the nonequivalence, the stability of α-helix of two block copolypeptides, (L -Ala)₂₀-(L -Glu)₂₀-(L -Phe) (designated as AEF) and (L -Glu)₂₀-(L -Ala)₂₀-(L -Phe) (EAF), in aqueous solution was investigated as a function of pH, temperature, and salt concentration by the measurement of the α-helical content using CD at 223 nm. The transition temperature, T_m, as a measure of the stability of the α-helix, decreased with increasing the salt concentration for EAF, while T_m increased for AEF. The results indicate that electrostatic interactions affect the nonequivalence of such helical stability. Thermodynamic quantities, ΔG, ΔH, and ΔS, of the thermal transition from random coil to α-helix were obtained by applying the curve-fitting method to the data. The major contribution to the effects of salts seems to be the entropic term, not the enthalpy term. This is unexpected, since the salt ions would weaken electrostatic interactions between ionized groups and the dipole along the helical axis, which affect the enthalpy term. In addition, the dependence of the electrostatic effect on the salt concentration is different for EAF and AEF. There fore, the nonequivalence cannot be accounted for by only the electrostatic effect, suggesting that it originates from some intrinsic property of the α-helix.     

A new method for predicting binding free energy between receptor and ligand

A practical method to estimate binding free energy, ΔG_bind, of a given ligand structure to the target receptor has been developed. The method assumes that ΔG_bind is given by the summation of intermolecular interaction energy, ΔG_inter, and partial desolvation energy, ΔG_desolv. ΔG_desolv is calculated from the buried surface area in the complex between the ligand and receptor, based on solvation energy, ΔG_solv, formulated by an equation which can be calibrated with observed values. Then, the method was applied to arabinose-binding protein (ABP) and dihydrofolate reductase (DHFR), after recalibrating the weights for ΔG_inter and each term of ΔG_desolv using observed ΔG_bind data for 29 known ligands to avidin (AV). The usefulness of our method was confirmed by the fact that correlation coefficients between the calculated and observed ΔG_bind's in AV, ABP and DHFR were 0.92, 0.77, and 0.88, whereas the corresponding values obtained by simple force field calculation were 0.79, 0.30, and 0.79, respectively. Further investigations to improve the method and validate the parameters are in progress. Proteins 33:62–73, 1998. © 1998 Wiley-Liss, Inc.     

Systematic investigation of interactions between papain and MPA-capped CdTe quantum dots

Fluorescent quantum dots (QDs) have been widely applied in biological and biomedical areas, but relatively little is known about the interaction of QDs with some natural enzymes. Herein, the interactions between 3-mercaptopropionic acid-capped CdTe QDs (MPA-QDs) and papain were systematically investigated by UV–Vis absorption spectra, fluorescence spectra and circular dichroism (CD) spectra under the physiological conditions. The fluorescence spectra results indicated that MPA-QDs quenched the fluorescence intensity of papain. The modified Stern–Volmer quenching constant K _a at different temperatures and the corresponding thermodynamic parameters ΔH, ΔG and ΔS were also calculated. The binding of MPA-QDs and papain is a result of the formation of QDs-papain complex and the electrostatic interactions play a major role in stabilizing the complex. The CD technique was further used to analyze the conformational changes of papain induced by MPA-QDs and the results indicated that the biological activity of papain was affected by MPA-QDs dramatically.     

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 K_D, k_on, k_off, 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 T_m 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 conformational transition and chain association of ι-carrageenan in aqueous solution: Calorimetric and chirooptical data

Isothermal microcalorimetry, differential scanning calorimetry (DSC), and chirooptical data obtained for ι-carrageenan in NaCl, LiCl, and NaI aqueous solutions are presented. The experiments have been performed as a function of concentration both for the polymer and for the simple salt as a cosolute. The experimental findings consistently show the occurrence of a salt-induced disorder-to-order transition. From microcalorimetric experiments the exothermic enthalpy of transition ΔH_tr is obtained as the difference between the theoretical, purely electrostatic ΔH_el enthalpy change and the actual mixing enthalpy ΔH_mix, measured when a ι-carrageenan salt-free solution at constant polymer concentration is mixed with a 1:1 electrolyte solution of variable concentration. In the case of added NaCl, the absolute values of enthalpy changes |ΔH_tr| are in good agreement with those obtained for the opposite process, at comparable polymer and salt concentrations, from DSC melting curves. The microcalorimetric results show that the negative maximum value of ΔH_tr corresponding to the interaction of Li⁺ counterion with ι-carrageenan polyion results to be significantly lower than the corresponding values obtained for Na⁺ counterion. At variance with the microcalorimetric data, chirooptical results show that the salt-induced disorder-to-order transition, occurring in the 0.02–0.2M salt concentration range, appears to be complete at a concentration of about 0.08–0.1M of the simple ion, irrespective of the polymer concentration and of the nature of added electrolyte. © 1998 John Wiley & Sons, Inc. Biopoly 45: 105–117, 1998     

Study on the interaction between a water-soluble dinuclear nickel complex and bovine serum albumin by spectroscopic techniques

The interaction of a water-soluble dinuclear nickel(II) complex, [Ni₂(EGTB)(CH₃CN)₄](ClO₄)₄·4H₂O (EGTB = ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetrakis(2-benzimidazoyl)) (1), and bovine serum albumin (BSA) was investigated under physiological conditions using fluorescence, synchronous fluorescence, UV–vis absorption and circular dichroism (CD). The experimental results suggested that the nickel(II) complex could bind to BSA with binding constant (K) ~ 10⁴ M^?1 and quench the intrinsic fluorescence of BSA through a static quenching mechanism. The thermodynamic parameters, ΔG°, ΔH°, and ΔS°, calculated at different temperatures, indicated that the binding reaction was spontaneous and electrostatic interactions played a major role in this association. Based on the number of binding sites, it was considered that one molecule of complex 1 could bind to a single site or two sites of the BSA molecule or the two binding modes coexisted. In view of the results of site marker competition experiments, the reactive sites of BSA to complex 1 mainly located in subdomain IIA (site I) and subdomain IIIA (site II) of BSA. Moreover, the binding distance, r, between donor (BSA) and acceptor (complex 1) was 5.13 nm according to Förster nonradiation energy transfer theory. Finally, as shown by the UV–vis absorption, synchronous fluorescence and CD, complex 1 could induce conformation and microenvironmental changes of BSA. The results obtained herein will be of biological significance in toxicology investigation and anticancer metallodrug design.     

Enantioselective DNA binding of iron(II) complexes of methyl-substituted phenanthroline

The enantioselective binding of [Fe(4,7-dmp)₃]²⁺ (dmp: 4,7-dimethyl-1,10-phenantroline) and [Fe(3,4,7,8-tmp)₃]²⁺ (tmp: 3,4,7,8-tetramethyl-1,10-phenanthroline) to calf-thymus DNA (ct-DNA) has been systematically studied by monitoring the circular dichroism (CD) spectral profile of the iron(II) complexes in the absence and presence of ct-DNA. The effect of salt concentration and temperature on the degree of enantioselectivity of the ct-DNA binding of the iron(II) complexes, i.e. the molar ratio of Δ- to Λ-enantiomer in the solution or vice versa has been rigorously evaluated. It is noticeable that Δ-[Fe(4,7-dmp)₃]²⁺ and Λ-[Fe(3,4,7,8-tmp)₃]²⁺ are preferentially bound to ct-DNA as reflected in their opposite CD spectral profiles. The preferential binding of the Λ-enantiomer of [Fe(3,4,7,8-tmp)₃]²⁺ to ct-DNA compared to that of the Δ-enantiomer is associated with the bulkiness of the ancillary ligands due to substitution of four hydrogen atoms in 1,10-phenanthroline for four methyl groups. The determination of enantiomeric inversion constant (K_inv) at various salt concentrations has revealed that the degree of enantioselectivity is salt concentration dependent, indicating that electrostatic interaction is involved in the enantioselective binding of the iron(II) complexes to ct-DNA. Although [Fe(4,7-dmp)₃]²⁺ and [Fe(3,4,7,8-tmp)₃]²⁺ exhibit an opposite pattern in the CD spectra, the degree of their enantioselectivity (K_inv) is not different from each other significantly. A thermodynamic study on the enantioselective binding of [Fe(4,7-dmp)₃]²⁺ to ct-DNA using the van’t Hoff plot of ln K_inv versus 1/T has demonstrated that the enthalpy change (ΔH°) in the inversion process from the Λ- to Δ-enantiomer of [Fe(4,7-dmp)₃]²⁺ ct-DNA is positive, indicating that the process is endothermic and thus entropically driven.     

Improving the affinity of naphthalene diimide ligand to telomeric DNA by incorporating Zn2+ ions into its dipicolylamine groups

N,N′-bis[3-[3-(2,2′-dipicolyl)methylaminopropyl]-methylaminopropyl]naphthalene-1,4,5,8-tetracarboxylic acid diimide, 1, and its complex with zinc ions, 2, were investigated against telomeric sequences, [TAGGG(TTAGGG)₃] and [AGGG(TTAGGG)₃], which reveal different G-quadruplex structures depending on the conditions. Spectrophotometric, SPR, and CD techniques revealed that both ligands showed large binding constants to hybrid-type G-quadruplexes formed in the presence of K⁺ ions. Moreover, 2 revealed higher affinity to investigated oligonucleotides suggesting that complex of naphthalene diimide derivative with Zn²⁺, comparing to 1, provided additional electrostatic or coordination interactions between positively charged zinc ions and condensed negative charged phosphate anions from G4 DNA.     

A calorimetric study of the binding of 2'-deoxyuridine-5'-phosphate and 5-fluoro-2'-deoxyuridine-5'-phosphate to thymidylate synthetase.

The thermodynamic parameters, ΔH′, ΔG′, and ΔS′, and the stoichiometry for the binding of the substrate 2′-deoxyuridine-5′-phosphate (dUMP) and the inhibitor 5-fluoro-2′-deoxyuridine-5′-phosphate (FdUMP) to Lactobacillus casei thymidylate synthetase (TSase) have been investigated using both direct calorimetric methods and gel filtration methods. The data obtained show that two ligand binding sites are available but that the binding of the second mole of dUMP is extremely weak. Binding of the first mole of dUMP can best be illustrated by dUMP + TSase + H⁺?(dUMP-TSase-H⁺). [1] The enthalpy, ΔH₁′, for reaction [1] was measured directly on a flow modification of a Beckman Model 190B microcalorimeter. Experiments in two different buffers (I = 0.10 m) show that ΔH₁′ = ?28 kJ mol^?1 and that 0.87 mol of protons enters into the reaction. Analysis of thermal titrations for reaction [1] indicates a free energy change of ΔG₁′ = ?30 kJ mol^?1 (K₁ = 1.7 × 10⁵ m^?1). From these parameters, ΔS₁′ was calculated to be +5 J mol^?1 degree^?1, showing that the reaction is almost totally driven by enthalpy changes. Gel filtration experiments show that at very high substrate concentrations, binding to a second site can be observed. Gel filtration experiments performed at low ionic strength (I = 0.05 m) reveal a stronger binding, with ΔG₁′ = ?35 kJ mol^?1 (K₁ = 1.2 × 10⁶ m^?1), suggesting that the forces driving the interaction are, in part, electrostatic. Addition of 2-mercaptoethanol (0.10 m) had the effect of slightly increasing the dUMP binding constant. Binding of FdUMP to TSase is best illustrated by 2FdUMP + TSase + n_HH⁺?FdUMP₂ ? TSase ? (H⁺)_nH. [2] The enthalpy for this reaction, ΔH₂, was also measured calorimetrically and found to be ?30 kJ mol^?1 with n_H = 1.24 at pH 7.4 Assuming two FdUMP binding sites per dimer as established by Galivan et al. [Biochemistry15, 356–362 (1976)] our calorimetric results indicate different binding energies for each site. Based on the binding data, a thermodynamic model is presented which serves to rationalize much of the confusing physical and chemical data characterizing thymidylate synthetase.     

Binding of berberine to bovine serum albumin: spectroscopic approach

Fluorescence spectroscopy in combination with UV–vis absorption spectroscopy was employed to investigate the binding of an important traditional medicinal herb berberine to bovine serum albumin (BSA) under the physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of BSA by berberine is a result of the formation of berberine–BSA complex. Fluorescence quenching constants were determined using the Stern–Volmer equation and Scatchard equation to provide a measure of the binding affinity between berberine and BSA. The results of thermodynamic parameters ΔG, ΔH, ΔS at different temperatures indicate that the electrostatic interactions play a major role for berberine–BSA association. Site marker competitive experiments indicated that the binding of berberine to BSA primarily took place in site II. Furthermore, the Effect of supramolecules to berberine–BSA system, and the distance r between donor (BSA) and acceptor (berberine) was obtained according to fluorescence resonance energy transfer (FRET).