Electrostatic contributions to the binding free energy of the lambdacI repressor to DNA

A model based on the nonlinear Poisson-Boltzmann (NLPB) equation is used to study the electrostatic contribution to the binding free energy of the lambdacI repressor to its operator DNA. In particular, we use the Poisson-Boltzmann model to calculate the pKa shift of individual ionizable amino acids upon binding. We find that three residues on each monomer, Glu34, Glu83, and the amino terminus, have significant changes in their pKa and titrate between pH 4 and 9. This information is then used to calculate the pH dependence of the binding free energy. We find that the calculated pH dependence of binding accurately reproduces the available experimental data over a range of physiological pH values. The NLPB equation is then used to develop an overall picture of the electrostatics of the lambdacI repressor-operator interaction. We find that long-range Coulombic forces associated with the highly charged nucleic acid provide a strong driving force for the interaction of the protein with the DNA. These favorable electrostatic interactions are opposed, however, by unfavorable changes in the solvation of both the protein and the DNA upon binding. Specifically, the formation of a protein-DNA complex removes both charged and polar groups at the binding interface from solvent while it displaces salt from around the nucleic acid. As a result, the electrostatic contribution to the lambdacI repressor-operator interaction opposes binding by approximately 73 kcal/mol at physiological salt concentrations and neutral pH. A variety of entropic terms also oppose binding. The major force driving the binding process appears to be release of interfacial water from the protein and DNA surfaces upon complexation and, possibly, enhanced packing interactions between the protein and DNA in the interface. When the various nonelectrostatic terms are described with simple models that have been applied previously to other binding processes, a general picture of protein/DNA association emerges in which binding is driven by the nonpolar interactions, whereas specificity results from electrostatic interactions that weaken binding but are necessary components of any protein/DNA complex.     

Electrostatic contributions to heat capacity changes of DNA-ligand binding

Significant heat capacity changes (DeltaCp) often accompany protein unfolding, protein binding, and specific DNA-ligand binding reactions. Such changes are widely used to analyze contributions arising from hydrophobic and polar hydration. Current models relate the magnitude of DeltaCp to the solvent accessible surface area (ASA) of the molecule. However, for many binding systems-particularly those involving non-peptide ligands-these models predict a DeltaCp that is significantly different from the experimentally measured value. Electrostatic interactions provide a potential source of heat capacity changes and do not scale with ASA. Using finite-difference Poisson-Boltzmann methods (FDPB), we have determined the contribution of electrostatics to the DeltaCp associated with binding for DNA binding reactions involving the ligands DAPI, netropsin, lexitropsin, and the lambda repressor binding domain.     

Comparison between antigen-antibody binding energies and interfacial free energies

Antigen-antibody binding energies derived from equilibrium data are compared with the binding energies resulting from the interfacial free energies obtained from contact angle measurements of antigens and antibodies. From these interfacial free energies two sorts of theoretical antigen-antibody binding energies can be derived, as well as the Hamaker constants for most antigen-antibody systems. For interaction in vacuo the Hamaker constants obtained are between 4 and 6 X 10(-13) ergs, while these constants for hydrated antigen antibody interactions are less than 10(-14) ergs. For interactions in vacuo, interfacial free energies yield binding energies (delta Fa) that lie between -120 and -140 ergs/cm2. For interactions in the aqueous phase (with interstitial water still present), much lower binding energies (delta Fb) are derived, of the order of -.01 and -1 ergs/cm2. In comparison, dextran-anti-dextran interactions show a binding energy derived from equilibrium data (delta Feq) of the order of -10 ergs/cm2. In general the equilibrium binding energies delta Feq of most antigen-antibody systems would vary between -1 and -20 ergs/cm2. The implications of this comparison are discussed in the light of the influence of residual water between antigenic determinant and antibody-active site, as well as in the light of the degree of perfection of fit between these sites.     

Electrostatic contributions to protein-protein binding affinities: application to Rap/Raf interaction

The challenge of evaluating absolute binding free energies of protein-protein complexes is addressed using the scaled Protein Dipoles Langevin Dipoles (PDLD/S) model in combination with the Linear Response Approximation (LRA). This is done by taking the complex between Rap1A (Rap) and the p21ras binding domain of c-Raf (Raf-RBD) (Nassar et al., Nature 375:554-560, 1995) as a model system. Several formulations and different thermodynamic cycles are explored taking advantage of the LRA method and considering the protein reorganization during complex formation. The performance of different approximations is examined by comparing the calculated and observed absolute binding energies for the native complex and some of its mutants. The evaluation of the contributions of individual residues to the binding free energy, which is referred to here as group contributions is also examined. Special attention is paid to the role of the "dielectric constant," epsilon(in) which is in fact a scaling factor that represents the contributions that are treated implicitly. It is found that explicit consideration of protein relaxation is crucial for obtaining reasonable results with small values of epsilon(in), but it is also found that such a treatment of protein-protein interactions is very challenging and does not always give stable results. This indicates that more advanced explicit calculations should be based on experimentally determined structures of both the complex and the isolated proteins. Nevertheless, it is demonstrated that the qualitative trend of the effect of mutations can be reproduced by considering the effect of protein reorganization implicitly, using epsilon(in) approximately 25 for ionized residues and epsilon(in) approximately 4 for polar residues. Thus, it is concluded that an explicit treatment of solvent relaxation (which is common to current continuum models) does not provide sufficient compensation for turning off the charges of ionized residues on the interaction surface of the Raf-RBD/Rap complex. Representing the missing contribution by large epsilon(in) can, of course, reproduce the observed effect of ionized residues, but now the contribution of uncharged residues will be largely underestimated. Regardless of these conceptual problems, it is established that a very simple nonrelaxed approach, where the relaxation of both the protein and the solvent are considered implicitly, can provide an effective qualitative way for evaluating group contributions, using large and small values for epsilon(in) of ionized and neutral residues, respectively. As much as the actual system studied is concerned we find that more residues than generally assumed play a role in Raf-RBD/Rap interaction. This includes residues that are not located at the protein-protein interaction surface. These residues contribute to the binding energy through direct charge-charge interaction without leading to drastic structural changes. The overall contribution of the surface residues is quite significant since Raf and Rap are positively and negatively charged, respectively, and their charges are distributed along the interaction site between the two proteins.     

Ultratight DNA binding of a new bisintercalating anthracycline antibiotic

Differential scanning calorimetry and absorption spectroscopy were used to characterize the interaction of the new bisintercalating anthracycline antibiotic, WP631, with DNA. The method of continuous variations revealed five distinct binding modes for WP631, corresponding to 6, 3, 1.3, 0.5, and 0.25 mol of base pairs (bp) per mole of ligand. The binding of one drug to 6 bp corresponds to the bisintercalative binding mode determined previously, and was the mode studied in detail. UV melting experiments and differential scanning calorimetry were used to measure the ultratight binding of WP631 to DNA. The binding constant for the interaction of WP631 with herring sperm DNA was determined to be 3.1 (+/- 0.2) x 10(11) M-1 at 20 degrees C. The large, favorable binding free energy of -15.3 kcal mol-1 was found to result from a large, negative enthalpic contribution of -30.2 kcal mol-1. DNA melting curves at different concentrations of WP631 were fitted to McGhee's model of DNA melting in the presence of ligands, yielding an independent estimate of DNA binding parameters. The salt dependence of the WP631 binding constant was examined, yielding a slope SK = delta (log K)/delta (log[Na+]) = 1.63. The observed salt dependence of the equilibrium constant, interpreted according to polyelectrolyte theory, indicates that there is a significant nonpolyelectrolyte contribution to the binding free energy. DNA melting studies using a homogeneous 214 bp DNA fragment showed that WP631 binds preferentially to the GC-rich region of the DNA.     

Dynamic contributions to the DNA binding entropy of the EcoRI and EcoRV restriction endonucleases

Molecular Dynamics simulations on DNA-EcoRI and DNA-EcoRV complexes suggest that the DNA within these complexes is significantly more ordered than free DNA. Similarly, both the protein and the DNA are more ordered in the specific (cognate) DNA-EcoRV complex than they are in the non-cognate DNA-protein complex, consistent with recently proposed analogies between protein folding and sequence-specific DNA-protein recognition. Analysis of the trajectories shows that the net entropy gain upon specific binding to be the result of opposing contributions. Solvent release, which increases entropy versus configurational terms (as measured by the magnitude of the atomic fluctuations), and collective terms from tight coupling between the motions of the protein and the DNA.     

Biosynthesis of anthracycline antibiotics by Streptomyces galilaeus. II. Structure of new anthracycline antibiotics obtained by microbial glycosidation and biological activity

New anthracycline antibiotics derived from epsilon-, gamma- and beta-rhodomycinones and epsilon-isorhodomycinone by the microbial glycosidation using an aclacinomycin-negative mutant, the strain KE303, of Streptomyces galilaeus MA144-M1 were studied to elucidate their structures and biological activities. These antibiotics were the products in which the anthracyclinones added as precursors were linked at C-7 or C-10 position with the same trisaccharide moiety (cinerulosyl-2-deoxyfucosyl-rhodosaminyl group) as in the parental antibiotic aclacinomycin A. In addition to antimicrobial activity, they exhibited the growth inhibition of cultured L1210 leukemia cells and the marked inhibition against DNA and RNA synthesis.     

Estimation of binding free energies for HIV proteinase inhibitors by molecular dynamics simulations

Absolute binding free energies for three inhibitors of HIV-1 proteinase were estimated from molecular dynamics simulations by a recently reported linear approximation procedure. The results were in fairly good agreement with experimental binding data. Two of the inhibitors were very similar and, for comparison, their relative free energies of binding were also calculated by free energy perturbation methods, giving virtually the same result. Effects of cut-off radii and charge states of the protein model were examined. The effects of pH on binding of one of the inhibitors were predicted.     

Specific binding of aminoglycoside antibiotics to RNA

BACKGROUND: Aminoglycoside antibiotics interfere with ribosomal protein synthesis and with intron splicing. Various lines of evidence suggest that RNA is the molecular target for aminoglycosides, but little is known about the recognition process. Is recognition of a particular aminoglycoside specific for certain RNA structures? If so, what are the rules for recognition? We have begun to investigate this problem by in vitro selection of RNA molecules that can specifically bind to the aminoglycoside antibiotic tobramycin. RESULTS: An RNA diversity library was used to select for sequences capable of binding to the aminoglycoside antibiotic tobramycin. After six cycles of selection, 82% of the RNA bound to tobramycin specifically. The selected RNA was reverse-transcribed into DNA, which was then cloned. At low selection stringency, an extremely large number of clones, on the order of 10(7), produced RNAs capable of binding tobramycin with Kds in the microM range (values similar to that observed for the binding of tobramycin to Escherichia coli ribosomes). Sequencing of 18 of the clones revealed no obvious consensus sequence. At higher selection stringencies (Kds in the nM range) only two consensus sequences for binding were observed. CONCLUSIONS: We have shown that RNA molecules can be readily selected that bind the aminoglycoside tobramycin. The RNAs that bind tobramycin with high affinity contain consensus binding regions that may be confined to predicted stem-loop structures. These studies open the way for understanding the basis of RNA-aminoglycoside recognition.     

Demethyl mutactimycins, new anthracycline antibiotics from Nocardia and Streptomyces strains

New anthracycline antibiotics 3'-O-demethyl mutactimycin (3) and 4-O,3'-O-didemethyl mutactimycin (4) were isolated from two actinomycetes strains, Nocardia transvalensis and Streptomyces sp. GW 60/1571. The chemical structures were elucidated by mass spectrometry and NMR spectroscopy. Antibiotic 3 displayed moderate antimicrobial activity against Gram-positive bacteria and cytotoxicity against P388, L1210 and HeLa tumor cells (IC50; 9.6, >25 and 20 microg/ml, respectively).     

Electrostatic and hydrophobic contributions to the folding mechanism of apocytochrome c driven by the interaction with lipid

In aqueous solution, while cytochrome c is a stably folded protein with a tightly packed structure at the secondary and tertiary levels, its heme-free precursor, apocytochrome c, shows all features of a structureless random coil. However, upon interaction with phospholipid vesicles or lysophospholipid micelles, apocytochrome c undergoes a conformational transition from its random coil in solution to an alpha-helical structure on association with lipid. The driving forces of this lipid-induced folding process of apocytochrome c were investigated for the interaction with various phospholipids and lysophospholipids. Binding of apocytochrome c to negatively charged phospholipid vesicles induced a partially folded state with approximately 85% of the alpha-helical structure of cytochrome c in solution. In contrast, in the presence of zwitterionic phospholipid vesicles, apocytochrome c remains a random coil, suggesting that negatively charged phospholipid headgroups play an important role in the mechanism of lipid-induced folding of apocytochrome c. However, negatively charged lysophospholipid micelles induce a higher content of alpha-helical structure than equivalent negatively charged diacylphospholipids in bilayers, reaching 100% of the alpha-helix content of cytochrome c in solution. Furthermore, micelles of lysolipids with the same zwitterionic headgroup of phospholipid bilayer vesicles induce approximately 60% of the alpha-helix content of cytochrome c in solution. On the basis of these results, we propose a mechanism for the folding of apocytochrome c induced by the interaction with lipid, which accounts for both electrostatic and hydrophobic contributions. Electrostatic lipid-protein interactions appear to direct the polypeptide to the micelle or vesicle surface and to induce an early partially folded state on the membrane surface. Hydrophobic interactions between nonpolar residues in the protein and the hydrophobic core of the lipid bilayer stabilize and extend the secondary structure upon membrane insertion.     

Transformation of cooperative free energies between ligation systems of hemoglobin: resolution of the carbon monoxide binding intermediates

A strategy has been developed for quantitatively "translating" the distributions of cooperative free energy between different oxygenation analogs of hemoglobin (Hb). The method was used to resolve the cooperative free energies of all eight carbon monoxide binding intermediates. These parameters of the FeCOHb system were determined by thermodynamic transformation of corresponding free energies obtained previously for all species of the Co/FeCO system, i.e., where cobalt-substituted hemes comprise the unligated sites [Speros, P. C., et al. (1991) Biochemistry 30, 7254-7262]. Using hybridized combinations of normal and cobalt-substituted Hb, ligation analog systems Co/FeX (X = CO, CN) were constructed and experimentally quantified. Energetics of cobalt-induced structural perturbation were determined for all species of both the "mixed metal" Co/Fe system and also the ligated Co/FeCN system. It was found that major energetic perturbations of the Co/Fe hybrid species originate from a pure cobalt substitution effect on the alpha subunits. These perturbations are transduced to the beta subunit within the same dimeric half-tetramer, resulting in alteration of the free energies for binding at the nonsubstituted (Fe) sites. Using the linkage strategy developed in this study along with the determined energetics of these couplings, the experimental assembly free energies for the Co/FeCO species were transformed into cooperative free energies of the 10 Fe/FeCO species. The resulting values were found to distribute according to predictions of a symmetry rule mechanism proposed previously [Ackers, G. K., et al. (1992) Science 255, 54-63]. Their distribution is consistent with accurate CO binding data of normal Hb [Perrella, M., et al. (1990b) Biophys. Chem. 37, 211-223] and also with accurate O2 binding data obtained under the same conditions [Chu, A. H., et al. (1984) Biochemistry 23, 604-617].     

Enzymatic and chemical footprinting of anthracycline antitumor antibiotics and related saccharide side chains

DNase I and three DNA chemical footprinting agents were used to compare the DNA binding properties of the anthracycline antitumor antibiotics daunomycin, aclacinomycin A, and ditrisarubicin B. These anthracyclines contain a tetracyclic chromophore which intercalates into DNA and a monosaccharide, trisaccharide, and two trisaccharide side chains, respectively. These side chains consist of between one and three 2,6-dideoxy, 1,4-diaxially linked sugars. Three chemical probes, fotemustine, dimethyl sulfate, 4-(2'-bromoethyl)phenol, and the enzymic probe DNase I were used in the footprinting experiments. The chemical probes provided a clear picture of the binding pattern at 37 degrees C and more detailed information than that obtained using the standard DNase I footprinting assay. All three anthracyclines showed preferred binding to 5'-GT-3' sequences in both the chemical and enzymatic footprinting. DNase I footprinting showed that the number of base pairs of DNA protected from cleavage increased with the number of saccharide groups present at particular sites and is consistent with DNA binding of the saccharide side chains. Alkylation of runs of guanine by fotemustine was inhibited by all three anthracyclines, while alkylation by dimethyl sulfate was enhanced for most guanines. The probe 4-(2'-bromoethyl)phenol showed that all three anthracyclines completely protected all of the adenines in the minor groove from alkylation, and enhanced major groove guanine alkylation was observed with aclacinomycin A, daunomycin, and, to a much lesser extent, ditrisarubicin B. These results are consistent with intercalation of the aglycone ring and binding of the rigid, hydrophobic saccharide side chains in the minor groove. Footprinting of four methyl glycosides related to the anthracyclines showed no evidence of DNA binding with any of the agents studied.