Reciprocal cooperative effects of multiple ligand binding to pyruvate kinase

The formation of multiple ligand complexes with muscle pyruvate kinase was measured in terms of dissociation constants and the standard free energies of formation were calculated. The binding of Mn2+ to the enzyme (KA = 55 +/- 5 X 10(-6) M; deltaF degrees = -5.75 +/- 0.05 kcal/mol) and to the enzyme saturated with phosphoenolpyruvate (conditional free energy) KA' = 0.8 +/- 0.4 X 10(-6) M; deltaF degrees = -8.22 +/- 0.34 kcal/mol) has been measured under identical conditions giving a free energy of coupling, delta(deltaF degrees) = -2.47 +/- 0.34 kcal/mol. Such a large negative free energy of coupling is diagnostic of a strong positively cooperative effect in ligand binding. The binding of the substrate phosphoenolpyruvate to free enzyme and the enzyme-Mn2+ complex was, by necessity, measured by different methods. The free energy of phosphoenolpyruvate binding to free enzyme (KS = 1.58 +/- 0.10 X 10(-4)M; deltaF degrees = -5.13 +/- 0.04 kcal/mol) and to the enzyme-Mn2+ complex (K3 = 0.75 +/- 0.10 X 10(-6)M; deltaF degrees = -8.26 +/- 0.07 kcal/mol) also gives a large negative free energy of coupling, delta(deltaF degrees) = -3.16 +/- 0.08 kcal/mol. Such a large negative value confirms reciprocal binding effects between the divalent cation and the substrate phosphoenolpyruvate. The binding of Mn2+ to the enzyme-ADP complex was also investigated and a free energy of coupling, delta(deltaF degrees) = -0.08 +/- 0.08 kcal/mol, was measured, indicative of little or no cooperativity in binding. The free energy of coupling with Mn2+ and pyruvate was measured as -1.52 +/- 0.14 kcal/mol, showing a significant amount of cooperativity in ligand binding but a substantially smaller effect than that observed for phosphoenolpyruvate binding. The magnitude of the coupling free energy may be related to the role of the divalent cation in the formation of the enzyme-substrate complexes. In the absence of the activating monovalent cation, the coupling free energies for phosphoenolpyruvate and pyruvate binding decrease by 40-60% and 25%, respectively, substantiating a role for the monovalent cation in the formation of enzyme-substrate complexes with phosphoenolpyruvate and with pyruvate.     

Glycogen phosphorylase inhibitors: a free energy perturbation analysis of glucopyranose spirohydantoin analogues

GP catalyzes the phosphorylation of glycogen to Glc-1-P. Because of its fundamental role in the metabolism of glycogen, GP has been the target for a systematic structure-assisted design of inhibitory compounds, which could be of value in the therapeutic treatment of type 2 diabetes mellitus. The most potent catalytic-site inhibitor of GP identified to date is spirohydantoin of glucopyranose (hydan). In this work, we employ MD free energy simulations to calculate the relative binding affinities for GP of hydan and two spirohydantoin analogues, methyl-hydan and n-hydan, in which a hydrogen atom is replaced by a methyl- or amino group, respectively. The results are compared with the experimental relative affinities of these ligands, estimated by kinetic measurements of the ligand inhibition constants. The calculated binding affinity for methyl-hydan (relative to hydan) is 3.75 +/- 1.4 kcal/mol, in excellent agreement with the experimental value (3.6 +/- 0.2 kcal/mol). For n-hydan, the calculated value is 1.0 +/- 1.1 kcal/mol, somewhat smaller than the experimental result (2.3 +/- 0.1 kcal/mol). A free energy decomposition analysis shows that hydan makes optimum interactions with protein residues and specific water molecules in the catalytic site. In the other two ligands, structural perturbations of the active site by the additional methyl- or amino group reduce the corresponding binding affinities. The computed binding free energies are sensitive to the preference of a specific water molecule for two well-defined positions in the catalytic site. The behavior of this water is analyzed in detail, and the free energy profile for the translocation of the water between the two positions is evaluated. The results provide insights into the role of water molecules in modulating ligand binding affinities. A comparison of the interactions between a set of ligands and their surrounding groups in X-ray structures is often used in the interpretation of binding free energy differences and in guiding the design of new ligands. For the systems in this work, such an approach fails to estimate the order of relative binding strengths, in contrast to the rigorous free energy treatment.     

Contributions to the binding free energy of ligands to avidin and streptavidin

The free energy of binding of a ligand to a macromolecule is here formally decomposed into the (effective) energy of interaction, reorganization energy of the ligand and the macromolecule, conformational entropy change of the ligand and the macromolecule, and translational and rotational entropy loss of the ligand. Molecular dynamics simulations with implicit solvation are used to evaluate these contributions in the binding of biotin, biotin analogs, and two peptides to avidin and streptavidin. We find that the largest contribution opposing binding is the protein reorganization energy, which is calculated to be from 10 to 30 kcal/mol for the ligands considered here. The ligand reorganization energy is also significant for flexible ligands. The translational/rotational entropy is 4.5-6 kcal/mol at 1 M standard state and room temperature. The calculated binding free energies are in the correct range, but the large statistical uncertainty in the protein reorganization energy precludes precise predictions. For some complexes, the simulations show multiple binding modes, different from the one observed in the crystal structure. This finding is probably due to deficiencies in the force field but may also reflect considerable ligand flexibility.     

Theoretical study of the ligand-CYP2B4 complexes: effect of structure on binding free energies and heme spin state

The molecular origins of temperature-dependent ligand-binding affinities and ligand-induced heme spin state conversion have been investigated using free energy analysis and DFT calculations for substrates and inhibitors of cytochrome P450 2B4 (CYP2B4), employing models of CYP2B4 based on CYP2C5(3LVdH)/CYP2C9 crystal structures, and the results compared with experiment. DFT calculations indicate that large heme-ligand interactions (ca. -15 kcal/mol) are required for inducing a high to low spin heme transition, which is correlated with large molecular electrostatic potentials (approximately -45 kcal/mol) at the ligand heteroatom. While type II ligands often contain oxygen and nitrogen heteroatoms that ligate heme iron, DFT results indicate that BP and MF heme complexes, with weak substrate-heme interactions (ca. -2 kcal/mol), and modest MEPS minima (>-35 kcal/mol) are high spin. In contrast, heme complexes of the CYP2B4 inhibitor, 4PI, the product of benzphetamine metabolism, DMBP, and water are low spin, have substantial heme-ligand interaction energies (<-15 kcal/mol) and deep MEPS minima (<-45 kcal/mol) near their heteroatoms. MMPBSA analysis of MD trajectories were made to estimate binding free energies of these ligands at the heme binding site of CYP2B4. In order to initially assess the realism of this approach, the binding free energy of 4PI inhibitor was computed and found to be a reasonable agreement with experiment: -7.7 kcal/mol [-7.2 kcal/mol (experiment)]. BP was determined to be a good substrate [-6.3 kcal/mol (with heme-ligand water), -7.3 kcal/mol (without ligand water)/-5.8 kcal/mol (experiment)], whereas the binding of MF was negligible, with only marginal binding binding free energy of -1.7 kcal/mol with 2-MF bound [-3.8 kcal/mol (experiment)], both with and without retained heme-ligand water. Analysis of the free energy components reveal that hydrophobic/nonpolar contributions account for approximately 90% of the total binding free energy of these substrates and are the source of their differential and temperature-dependent CYP2B4 binding. The results indicate the underlying origins of the experimentally observed differential binding affinities of BP and MF, and indicate the plausibility of the use of models derived from moderate sequence identity templates in conjunction with approximate free energy methods in the estimation of ligand-P450 binding affinities.     

Analysis of the binding energies of testosterone, 5alpha-dihydrotestosterone,androstenedione and dehydroepiandrosterone sulfate with an antitestosterone antibody

Molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) free energy calculations were used to study the binding of testosterone (TES), 5alpha-dihydrotestosterone (5ADHT), androstenedione (AND), and dehydroepiandrosterone sulfate (DHEAS) to the monoclonal antitestosterone antibody 3-C(4)F(5). The relative binding free energy of TES and AND was also calculated with free energy perturbation (FEP) simulations. The antibody 3-C(4)F(5) has a relatively high affinity (3 x 10(8) M(-1)) and on overall good binding profile for testosterone but its cross-reactivity with DHEAS has been the main reason for the failure to use this antibody in clinical immunoassays. The relative binding free energies obtained with the MM-PBSA method were 1.5 kcal/mol for 5ADHT, 3.8 kcal/mol for AND, and 4.3 kcal/mol for DHEAS, as compared to TES. When a water molecule of the ligand binding site, observed in the antibody-TES crystal structure, was explicitly included in MM-PBSA calculations, the relative binding energies were 3.4, 4.9, and 5.4 kcal/mol for 5ADHT, AND, and DHEAS, respectively. The calculated numbers are in correct order but larger than the corresponding experimental energies of 1.3, 1.5, and 2.6 kcal/mol, respectively. The fact that the MM-PBSA method reproduced the relative binding free energies of DHEAS, a steroid having a negatively charged sulfate group, and the neutrally charged TES, 5ADHT, and AND in satisfactory agreement with experiment shows the robustness of the method in predicting relative binding affinities. The 800-ps FEP simulations predicted that the antibody 3-C(4)F(5) binds TES 1.3 kcal/mol tighter than AND. Computational mutagenesis of selected amino acid residues of the ligand binding site revealed that the lower affinities of AND and DHEAS as compared to TES are due to a combined effect of several residues, each contributing a small fraction to the tighter binding of TES. An exception to this is Tyr99H, whose mutation to Ala lowered the binding of DHEAS 0.7 kcal/mol more than the binding of TES. This is probably due to the hydrogen bonding interaction formed between the OH group of Tyr99H and the sulfate group of DHEAS. Computational mutagensis data also showed that the affinity of the steroids to the antitestosterone antibody 3-C(4)F(5) would be enhanced if Trp47H were repositioned so that it would make more extensive contacts with the bound ligands. In addition, the binding of steroids to antitestosterone, antiprogesterone, and antiestradiol antibodies is discussed.     

Empirical calculation of the relative free energies of peptide binding to the molecular chaperone DnaK

We describe a methodology to calculate the relative free energies of protein-peptide complex formation. The interaction energy was decomposed into nonpolar, electrostatic and entropic contributions. A free energy-surface area relationship served to calculate the nonpolar free energy term. The electrostatic free energy was calculated with the finite difference Poisson-Boltzmann method and the entropic contribution was estimated from the loss in the conformational entropy of the peptide side chains. We applied this methodology to a series of DnaK*peptide complexes. On the basis of the single known crystal structure of the peptide-binding domain of DnaK with a bound heptapeptide, we modeled ten other DnaK*heptapeptide complexes with experimentally measured K(d) values from 0.06 microM to 11 microM, using molecular dynamics to refine the structures of the complexes. Molecular dynamic trajectories, after equilibration, were used for calculating the energies with greater accuracy. The calculated relative binding free energies were compared with the experimentally determined free energies. Linear scaling of the calculated terms was applied to fit them to the experimental values. The calculated binding free energies were between -7.1 kcal/mol and - 9.4 kcal/mol with a correlation coefficient of 0.86. The calculated nonpolar contributions are mainly due to the central hydrophobic binding pocket of DnaK for three amino acid residues. Negative electrostatic fields generated by the protein increase the binding affinity for basic residues flanking the hydrophobic core of the peptide ligand. Analysis of the individual energy contributions indicated that the nonpolar contributions are predominant compared to the other energy terms even for peptides with low affinity and that inclusion of the change in conformational entropy of the peptide side chains does not improve the discriminative power of the calculation. The method seems to be useful for predicting relative binding energies of peptide ligands of DnaK and might be applicable to other protein-peptide systems, particularly if only the structure of one protein-ligand complex is available.     

Site-specific enthalpic regulation of DNA transcription at bacteriophage lambda OR.

Binding of cI repressor to DNA fragments containing the three specific binding sites of the right operator (OR) of bacteriophage lambda was studied in vitro over the temperature range 5-37 degrees C by quantitative footprint titration. The individual-site isotherms, obtained for binding repressor dimers to each site of wild-type OR and to appropriate mutant operator templates, were analyzed for the Gibbs energies of intrinsic binding and pairwise cooperative interactions. It is found that dimer affinity for each of the three sites varies inversely with temperature, i.e., the binding reactions are enthalpy driven, unlike many protein-DNA reactions. By contrast, the magnitude of the pairwise cooperativity terms describing interaction between adjacently site-bound repressor dimers is quite small. This result in combination with the recent finding that repressor monomer-dimer assembly is highly enthalpy driven (with delta H degrees = -16 kcal mol-1) [Koblan, K. S., & Ackers, G. K. (1991) Biochemistry 30, 7817-7821] indicates that the associative contacts between site-bound repressors that mediate cooperativity are unlikely to be the same as those responsible for dimerization. The intrinsic binding enthalpies for all three sites are negative (exothermic) and nearly temperature-invariant, indicating no heat capacity changes on the scale of those inferred in other protein-DNA systems. However, the three operator sites are affected differentially by temperature: the intrinsic binding free energies for sites OR1 and OR3 change in parallel over the entire range, delta H0OR1 = -23.3 +/- 4.0 kcal mol-1 and delta H0OR3 = -22.7 +/- 1.2 kcal mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free energy simulations and MM-PBSA analyses on the affinity and specificity of steroid binding to antiestradiol antibody

Antiestradiol antibody 57-2 binds 17beta-estradiol (E2) with moderately high affinity (K(a) = 5 x 10(8) M(-1)). The structurally related natural estrogens estrone and estriol as well synthetic 17-deoxy-estradiol and 17alpha-estradiol are bound to the antibody with 3.7-4.9 kcal mol(-1) lower binding free energies than E2. Free energy perturbation (FEP) simulations and the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method were applied to investigate the factors responsible for the relatively low cross-reactivity of the antibody with these four steroids, differing from E2 by the substituents of the steroid D-ring. In addition, computational alanine scanning of the binding site residues was carried out with the MM-PBSA method. Both the FEP and MM-PBSA methods reproduced the experimental relative affinities of the five steroids in good agreement with experiment. On the basis of FEP simulations, the number of hydrogen bonds formed between the antibody and steroids, which varied from 0 to 3 in the steroids studied, determined directly the magnitude of the steroid-antibody interaction free energies. One hydrogen bond was calculated to contribute about 3 kcal mol(-1) to the interaction energy. Because the relative binding free energies of estrone (two antibody-steroid hydrogen bonds), estriol (three hydrogen bonds), 17-deoxy-estradiol (no hydrogen bonds), and 17alpha-estradiol (two hydrogen bonds) are close to each other and clearly lower than that of E2 (three hydrogen bonds), the water-steroid interactions lost upon binding to the antibody make an important contribution to the binding free energies. The MM-PBSA calculations showed that the binding of steroids to the antiestradiol antibody is driven by van der Waals interactions, whereas specificity is solely due to electrostatic interactions. In addition, binding of steroids to the antiestradiol antibody 57-2 was compared to the binding to the antiprogesterone antibody DB3 and antitestosterone antibody 3-C4F5, studied earlier with the MM-PBSA method.     

Mapping the energetics of water-protein and water-ligand interactions with the "natural" HINT forcefield: predictive tools for characterizing the roles of water in biomolecules

The energetics and hydrogen bonding pattern of water molecules bound to proteins were mapped by analyzing structural data (resolution better than 2.3A) for sets of uncomplexed and ligand-complexed proteins. Water-protein and water-ligand interactions were evaluated using hydropatic interactions (HINT), a non-Newtonian forcefield based on experimentally determined logP(octanol/water) values. Potential water hydrogen bonding ability was assessed by a new Rank algorithm. The HINT-derived binding energies and Ranks for second shell water molecules were -0.04 kcal mol(-1) and 0.0, respectively, for first shell water molecules -0.38 kcal mol(-1) and 1.6, for active site water molecules -0.45 kcal mol(-1) and 2.3, for cavity water molecules -0.55 kcal mol(-1) and 3.3, and for buried water molecules -0.56 kcal mol(-1) and 4.4. For the last four classes, similar energies indicate that internal and external water molecules interact with protein almost equally, despite different degrees of hydrogen bonding. The binding energies and Ranks for water molecules bridging ligand-protein were -1.13 kcal mol(-1) and 4.5, respectively. This energetic contribution is shared equally between protein and ligand, whereas Rank favors the protein. Lastly, by comparing the uncomplexed and complexed forms of proteins, guidelines were developed for prediction of the roles played by active site water molecules in ligand binding. A water molecule with high Rank and HINT score is unlikely to make further interactions with the ligand and is largely irrelevant to the binding process, while a water molecule with moderate Rank and high HINT score is available for ligand interaction. Water molecule displaced for steric reasons were characterized by lower Rank and HINT score. These guidelines, tested by calculating HINT score and Rank for 50 water molecules bound in the active site of four uncomplexed proteins (for which the structures of the liganded forms were also available), correctly predicted the ultimate roles (in the complex) for 76% of water molecules. Some failures were likely due to ambiguities in the structural data.     

The molecular code for hemoglobin allostery revealed by linking the thermodynamics and kinetics of quaternary structural change. 2. Cooperative free energies of (alphaFeCObetaFe)2 and (alphaFebetaFeCO)2 T-state tetramers

Ligand photodissociation experiments are used to measure the prephotolysis equilibria between doubly liganded R and T quaternary conformers of the symmetric Fe-Co HbCO hybrids, (alpha(FeCO)beta(Co))(2) and (alpha(Co)beta(FeCO))(2). The free energies obtained from these data are used to calculate the cooperative free energies of the (alpha(FeCO)beta(Fe))(2) and (alpha(Fe)beta(FeCO))(2) intermediate CO-ligation states of normal hemoglobin in the T conformation, quantities important to the evaluation of current models of cooperativity. The symmetry rule model, incorporating sequential cooperativity of T-state ligand binding within an alphabeta dimer in addition to the traditional two-state cooperativity of the tetramer, predicts a larger free energy penalty for disturbing both dimers in a doubly liganded T tetramer than would be expected in the two-state model as currently formulated. (Cooperative energy penalties are simply proportional to the number of tetramer-bound ligands in the traditional two-state model.) The value found here for the energies of doubly liganded T microstates in which both dimers are perturbed, 7.9 +/- 0.3 kcal/mol, is consistent with the symmetry rule model but significantly higher than that expected (5-6 kcal/mol) in the two-state model of cooperativity.     

Predicting Ligand Binding Affinity with Alchemical Free Energy Methods in a Polar Model Binding Site

We present a combined experimental and modeling study of organic ligand molecules binding to a slightly polar engineered cavity site in T4 lysozyme (L99A/M102Q). For modeling, we computed alchemical absolute binding free energies. These were blind tests performed prospectively on 13 diverse, previously untested candidate ligand molecules. We predicted that eight compounds would bind to the cavity and five would not; 11 of 13 predictions were correct at this level. The RMS error to the measurable absolute binding energies was 1.8 kcal/mol. In addition, we computed “relative” binding free energies for six phenol derivatives starting from two known ligands: phenol and catechol. The average RMS error in the relative free energy prediction was 2.5 kcal/mol (phenol) and 1.1 kcal/mol (catechol). To understand these results at atomic resolution, we obtained x-ray co-complex structures for nine of the diverse ligands and for all six phenol analogs. The average RMSD of the predicted pose to the experiment was 2.0 Å (diverse set), 1.8 Å (phenol-derived predictions), and 1.2 Å (catechol-derived predictions). We found that predicting accurate affinities and rank-orderings required near-native starting orientations of the ligand in the binding site. Unanticipated binding modes, multiple ligand binding, and protein conformational change all proved challenging for the free energy methods. We believe that these results can help guide future improvements in physics-based absolute binding free energy methods.     

Kinetic linked-function analysis of the multiligand interactions on Mg(2+)-activated yeast pyruvate kinase.

The multiligand interactions governing the allosteric response of Mg(2+)-activated yeast pyruvate kinase (YPK) during steady-state turnover were quantitated by kinetic linked-function analysis. The substrate, PEP, the enzyme-bound divalent metal, Mg(2+), and the allosteric effector, FBP, positively influence each other's interaction with the enzyme in the presence of saturating concentrations of the second substrate, MgADP. The presence of Mg(2+) enhances the interaction of PEP and of FBP with YPK by -2.0 and -1.0 kcal/mol, respectively. The simultaneous interaction of PEP, Mg(2+), and FBP with YPK is favored by -4.1 kcal/mol over the sum of their independent binding free energies. The coupling free energies measured for Mg(2+)-activated YPK are weaker than the corresponding coupling free energies measured for Mn(2+)-activated YPK [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803], but are consistent with results of thermodynamic measurements with the Mg(2+)-YPK complex [Bollenbach, T. J., and Nowak, T. (2001) Biochemistry 36, 13088-13096]. A comparison of ligand binding data measured by kinetic and thermodynamic linked-function analyses reveals that the MgADP complex modulates both the binding of the other three ligands and the two- and three-ligand coupling interactions between the other three ligands. Enzyme-bound Mg(2+) does not influence the homotropic cooperativity in PEP binding to YPK. It is the MgADP complex that induces homotropic cooperativity in PEP binding. It is the enzyme-bound Mn(2+) that induces homotropic binding of PEP with Mn(2+)-activated YPK. These results lend support to the hypothesis that divalent metals modulate the interactions of ligands on YPK and that divalent metals play a role in regulation of the glycolytic pathway.     

Influence of Glu-376 --> Gln mutation on enthalpy and heat capacity changes for the binding of slightly altered ligands to medium chain acyl-CoA dehydrogenase

We showed that the alpha-CH(2) --> NH substitution in octanoyl-CoA alters the ground and transition state energies for the binding of the CoA ligands to medium-chain acyl-CoA dehydrogenase (MCAD), and such an effect is caused by a small electrostatic difference between the ligands. To ascertain the extent that the electrostatic contribution of the ligand structure and/or the enzyme site environment modulates the thermodynamics of the enzyme-ligand interaction, we undertook comparative microcalorimetric studies for the binding of 2-azaoctanoyl-CoA (alpha-CH(2) --> NH substituted octanoyl-CoA) and octenoyl-CoA to the wild-type and Glu-376 --> Gln mutant enzymes. The experimental data revealed that both enthalpy (DeltaH degrees ) and heat capacity changes (DeltaC(p) degrees ) for the binding of 2-azaoctanoyl-CoA (DeltaH degrees (298) = -21.7 +/- 0.8 kcal/mole, DeltaC(p) degrees = -0.627 +/- 0.04 kcal/mole/K) to the wild-type MCAD were more negative than those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -17.2 +/- 1.6 kcal/mole, DeltaC(p) degrees = -0.526 +/- 0.03 kcal/mole/K). Of these, the decrease in the magnitude of DeltaC(p) degrees for the binding of 2-azaoctanoyl-CoA (vis-à-vis octenoyl-CoA) to the enzyme was unexpected, because the former ligand could be envisaged to be more polar than the latter. To our further surprise, the ligand-dependent discrimination in the above parameters was completely abolished on Glu-376 --> Gln mutation of the enzyme. Both DeltaH degrees and DeltaC(p) degrees values for the binding of 2-azaoctanoyl-CoA (DeltaH degrees (298) = -13.3 +/- 0.6 kcal/mole, DeltaC(p) degrees = -0.511 +/- 0.03 kcal/mole/K) to the E376Q mutant enzyme were found to be correspondingly identical to those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -13.2 +/- 0.6 kcal/mole, DeltaC(p) degrees = -0.520 +/- 0.02 kcal/mole/K). However, in neither case could the experimentally determined DeltaC(p) degrees values be predicted on the basis of the changes in the water accessible surface areas of the enzyme and ligand species. Arguments are presented that the origin of the above thermodynamic differences lies in solvent reorganization and water-mediated electrostatic interaction between ligands and enzyme site groups, and such interactions are intrinsic to the molecular basis of the enzyme-ligand complementarity.     

High free energy of lipid/protein interaction in biological membranes

The free energy of lipid/protein interaction in biological membranes is still unknown although extensive partitioning and modelling studies have revealed many partial energetic increments. Multiple site binding kinetics are now applied to four well-studied functional membrane proteins, and mean free energy values (+/-S.D.) of -4.23+/-0.49 kcal/mol for single lipid binding sites and of -89.7+/-35.4 kcal/mol for complete lipid substitution are obtained. These high free energy values point to an important bioenergetic role of lipid/protein interaction in membrane functions.     

Thermodynamic, spectroscopic, and equilibrium binding studies of DNA sequence context effects in four 40 base pair deoxyoligonucleotides

Effects of different end sequences on melting, circular dichroism spectra (CD), and enzyme binding properties were investigated for four 40 base pair, non-self-complementary duplex DNA oligomers. The center sequences of these oligoduplexes have either of two 22 base pair modules flanked on both sides by sequences differing in AT content. Temperature-induced melting transitions monitored by differential scanning calorimetry (DSC) and ultraviolet absorbance were measured for the six duplexes in buffered 115 mM Na(+) solutions. Values of the melting transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal), were obtained directly from DSC experiments. Melting transition parameters, DeltaH(vH) and DeltaS(vH), were also estimated from a van't Hoff analysis of optical melting curves collected as a function of DNA concentration, assuming that the melting transition is two-state. Melting free energies (20 degrees C) evaluated from DSC melting experiments on the four duplex DNAs ranged from -52.2 to -77.5 kcal/mol. Free energies based on the van't Hoff analysis were -37.9 to -58.8 kcal/mol. Although the values are different, trends in the melting free energies of the four duplex DNAs as a function of sequence were identical in both DSC and optical analyses. Subject to several assumptions, values for the initiation free energy were estimated for each duplex, defined as DeltaG(int) = DeltaG(cal) - DeltaG(pred), where DeltaG(cal) is the experimental free energy at 20 degrees C determined from the experimentially measured values of the transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal). The predicted free energy of the sequence, DeltaG(pred)(20 degrees C), is obtained using published nearest-neighbor sequence stability values. For three of the four duplexes, values of DeltaG(int) are essentially nil. In contrast, the duplex with 81.8% GC has a considerably higher estimate of DeltaG(int) = 7.1 kcal/mol. The CD spectra for the six duplexes collected over the wavelength range from 200 to 320 nm are also sequence-dependent. Factor analysis of the CD spectra by singular value decomposition revealed that the experimental CD spectra could be reconstructed from linear combinations of two minor and one major subspectra. Changes in the coefficients of the major subspectrum for different sequences reflect incremental sequence-dependent variations of the CD spectra. Equilibrium binding by BamHI restriction endonuclease to the 40 base pair DNAs whose central eight base pairs contain the recognition sequence for BamHI restriction enzyme bounded by A.T base pairs, 5'-A-GGATCC-A-3' was investigated. Binding assays were performed by titering BamHI against a constant concentration of each of the duplex DNA substrates, in the absence of Mg(2+), followed by analysis by gel retardation. Under the conditions employed, the enzyme binds but does not cleave the DNAs. Results of the assays revealed two binding modes with retarded gel mobilities. Binding isotherms for the fraction of bound DNA species versus enzyme concentration for each binding mode were constructed and analyzed with a simple two-step equilibrium binding model. This analysis provided semiquantitative estimates on the equilibrium binding constants for BamHI to the four DNAs. Values obtained for the binding constants varied only 7-fold and ranged from 6 x 10(-)(8) to 42 x 10(-)(8) M, with binding free energies from -8.6 to -9.7 (+/- 0.2) kcal/mol depending on the sequence that flanks the enzyme binding site. Unlike what was found earlier in binding studies of the 22 base pair duplexes that constitute the core modules of the present 40-mers [Riccelli, P. V., Vallone, P. M., Kashin, I., Faldasz, B. D., Lane, M. J., and Benight, A. S. (1999) Biochemistry 38, 11197-11208], no obvious relationship between binding and stability was found for these longer DNAs. Apparently, effects of flanking sequence stability on restriction enzyme binding may only be measurable in very short duplex deoxyoligonucl     

N-acetylcolchinol O-methyl ether and thiocolchicine, potent analogs of colchicine modified in the C ring. Evaluation of the mechanistic basis for their enhanced biological properties

Two colchicine analogs with modifications only in the C ring are better inhibitors than colchicine of cell growth and tubulin polymerization. Radiolabeled thiocolchicine (with a thiomethyl instead of a methoxy group at position C-10) and N-acetylcolchinol O-methyl ether (NCME) (with a methoxy-substituted benzenoid instead of the methoxy-substituted tropone C ring) were prepared for comparison with colchicine. Scatchard analysis indicated a single binding site with KD values of 1.0-2.3 microM. Thiocolchicine was bound 2-4 times as rapidly as colchicine, but the activation energies of the reactions were nearly identical (18 kcal/mol for colchicine, 20 kcal/mol for thiocolchicine). NCME bound to tubulin in a biphasic reaction. The faster phase was 60 times as fast as colchicine binding at 37 degrees C, and a substantial reaction occurred at 0 degrees C. The rate of the faster phase of NCME binding changed relatively little as a function of temperature, so the activation energy was only 7.0 kcal/mol. Dissociation reactions were also evaluated, and at 37 degrees C the half-lives of the tubulin-drug complexes were 11 min for NCME, 24 h for thiocolchicine, and 27 h for colchicine. Relative dissociation rates as a function of temperature varied little among the drug complexes. Activation energies for the dissociation reactions were 30 kcal/mol for thiocolchicine, 27 kcal/mol for NCME, and 24 kcal/mol for colchicine. Comparison of the activation energies of association and dissociation yielded free energies for the binding reactions of -20 kcal/mol for NCME, -10 kcal/mol for thiocolchicine, and -6 kcal/mol for colchicine. The greater effectiveness of NCME and thiocolchicine as compared with colchicine in biological assays probably derives from their more rapid binding to tubulin and the lower free energies of their binding reactions.     

Use of binding site neighbor-effect parameters to evaluate the interactions between adjacent ligands on a linear lattice. Effects on ligand-lattice association.

A method using binding site "neighbor-effect" parameters (NEPs) is introduced to evaluate the effects of interaction between adjacent ligands on their binding to an infinite linear lattice. Binding site overlap is also taken into account. This enables the conditional probability approach of McGhee & von Hippel to be extended to more complex situations. The general equation for the isotherm is v/LF = SFKF, where v is the ratio of bound ligands to lattice residues, LF is the free ligand concentration, SF is the fraction of binding sites that are free, and KF is the average association constant of a free site. Solutions are derived for three cases: symmetric ligands, and asymmetric ligands on isotropic or anisotropic lattices. For symmetric ligands there is one NEP, E, which is the ratio of the average binding affinity of a free site if the status of the lattice residue neighboring one end of the site is unspecified (left to chance) to the affinity when this residue is free (holding the other neighbor constant). Thus KF is KE2, where K is the affinity of an isolated site. If a site is n residues long, SF is f ffn-1, where f = 1 - nv is the fraction of residues that are free and ff is the conditional probability that a free residue is bordered on a given side by another free residue. The expression for ff is 1/(1 + x/E), where x is v/f, E is (1 - x + [(1 - x)2 + 4x omega]1/2)/2, and omega is the co-operativity parameter. The binding of asymmetric ligands to an isotropic lattice is described by two NEPs; the last case involves four NEPs and a bound ligand orientation parameter. For each case, the expected length distribution of clusters of bound ligands can be calculated as a function of v. When Scatchard plots with the same intercepts and initial slope are compared, it is found that ligand asymmetry lowers the isotherm (relative to the corresponding symmetric ligand isotherm), whereas lattice anisotrophy raises it.     

Modeling implicit reorganization in continuum descriptions of protein-protein interactions

The determination of free energies that govern protein-protein recognition is essential for a detailed molecular understanding of biological specificity. Continuum models of macromolecular interactions, in which the solvent is treated by an implicit representation and the proteins are treated semi-microscopically, are computationally tractable for estimating free energies, yet many questions remain concerning their accuracy. This article reports a continuum analysis of the free-energy changes underlying the binding of 31 interfacial alanine substitutions of two complexes of the antihen egg white lysozyme (HEL) antibody D1.3 bound with HEL or the antibody E5.2. Two implicit schemes for modeling the effects of protein and solvent relaxation were examined, in which the protein environment was treated as either homogeneous with a "protein dielectric constant" of epsilon(p) = 4 or inhomogeneous, with epsilon(p) = 4 for neutral residues and epsilon(p) = 25 for ionized residues. The results showed that the nonuniform dielectric model reproduced the experimental differences better, with an average absolute error of +/-1.1 kcal/mol, compared with +/-1.4 kcal/mol for the uniform model. More importantly, the error for charged residues in the nonuniform model is +/-0.8 kcal/mol and is nearly half of that corresponding to the uniform model. Several substitutions were clearly problematic in determining qualitative trends and probably required explicit structural reorganization at the protein-protein interface.