Counterion Release Research Articles

The energetics of HMG box interactions with DNA: thermodynamics of the DNA binding of the HMG box from mouse sox-5

The energetics of the Sox-5 HMG box interaction with DNA duplexes, containing the recognition sequence AACAAT, were studied by fluorescence spectroscopy, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). Fluorescence titration showed that the association constant of this HMG box with the duplexes is of the order 4×10 7 M −1, increasing somewhat with temperature rise, i.e. the Gibbs energy is −40 kJ mol −1 at 5 °C, decreasing to −48 kJ mol −1 at 32 °C. ITC measurements of the enthalpy of association over this temperature range showed an endothermic effect below 17 °C and an exothermic effect above, suggesting a heat capacity change on binding of about −4 kJ K −1 mol −1, a value twice larger than expected from structural considerations. A straightforward interpretation of ITC data in heat capacity terms assumes, however, that the heat capacities of all participants in the association reaction do not change over the considered temperature range. Our previous studies showed that over the temperature range of the ITC experiments the HMG box of Sox-5 starts to unfold, absorbing heat and the heat capacities of the DNA duplexes also increase significantly. These heat capacity effects differ from that of the DNA/Sox-5 complex. Correcting the ITC measured binding enthalpies for the heat capacity changes of the components and complex yielded the net enthalpies which exhibit a temperature dependence of about −2 kJ K −1 mol −1, in good agreement with that predicted on the basis of dehydration of the protein-DNA interface. Using the derived heat capacity change and the enthalpy and Gibbs energy of association measured at 5 °C, the net enthalpy and entropy of association of the fully folded HMG box with the target DNA duplexes was determined over a broad temperature range. These functions were compared with those for other known cases of sequence specific DNA/protein association. It appears that the enthalpy and entropy of association of minor groove binding proteins are more positive than for proteins binding in the major groove. The observed thermodynamic characteristics of protein binding to the A+T-rich minor groove of DNA might result from dehydration of both polar and non-polar groups at the interface and release of counterions. The expected entropy of dehydration was calculated and found to be too large to be compensated by the negative entropy of reduction of translational/rotational freedom. This implies that DNA/HMG box association proceeds with significant decrease of conformational entropy, i.e. reduction in conformational mobility.

Phase Diagram, Stability, and Overcharging of Lamellar Cationic Lipid–DNA Self-Assembled Complexes

Cationic lipid–DNA (CL-DNA) complexes comprise a promising new class of synthetic nonviral gene delivery systems. When positively charged, they attach to the anionic cell surface and transfer DNA into the cell cytoplasm. We report a comprehensive x-ray diffraction study of the lamellar CL-DNA self-assemblies as a function of lipid composition and lipid/DNA ratio, aimed at elucidating the interactions determining their structure, charge, and thermodynamic stability. The driving force for the formation of charge-neutral complexes is the release of DNA and lipid counterions. Negatively charged complexes have a higher DNA packing density than isoelectric complexes, whereas positively charged ones have a lower packing density. This indicates that the overcharging of the complex away from its isoelectric point is caused by changes of the bulk structure with absorption of excess DNA or cationic lipid. The degree of overcharging is dependent on the membrane charge density, which is controlled by the ratio of neutral to cationic lipid in the bilayers. Importantly, overcharged complexes are observed to move toward their isoelectric charge-neutral point at higher concentration of salt co-ions, with positively overcharged complexes expelling cationic lipid and negatively overcharged complexes expelling DNA. Our observations should apply universally to the formation and structure of self-assemblies between oppositely charged macromolecules.

Comment on: 'The entropy cost of protein association'.

An understanding of the origin of the entropy loss involved in association processes (protein‐protein or protein‐ligand) is important for an interpretation of the many equilibria that play a role in biology. Although the total entropy change on association can be determined by measuring the transition temperature and associated enthalpy change (Privalov et al., 1995), interpretation of the results is hindered by the fact that there are several partially cancelling contributions [Tidor and Karplus, 1993 (TK)]. The loss of three translations and three (two for a linear molecule) rotations is entropically unfavorable. This can be offset by favorable entropic contributions, including those that arise from the hydrophobic effect, changes in the protonation states, solvent and counter ion release, as well as the presence of six (five for a linear molecule) new vibrational degrees of freedom. Because the translational and rotational contributions to the entropy of binding appear to be very simple to calculate, they have often been discussed. However, there are no direct measurements of their contribution to binding and theoretical estimates vary by more than an order of magnitude. A recent paper by Tamura and Privalov (TP) (1997) purports to make a direct measurement of the translational/rotational entropy contribution to the formation of a dimer of the Streptomyces subtilisin inhibitor (SSI). TP studied the temperature-induced unfolding of the wild-type dimer and of a mutant (D83C), with a disulfide cross-link between the subunits, by differential scanning calorimetry; the measurements were made as a function of concentration. When normalized to the same temperature, the entropy of unfolding of the native SSI dimer adjusted t oa1M standard state was found to be approximately the same as that of the cross-linked mutant dimer; the measured difference was ‐5 6 4 cal/mol·K. This result is very different from that obtained in an earlier calorimetric study of the same system by Tamura et al. (1994), who found a value of about ‐100 cal/mol·K for the difference of the entropies of unfolding of the two systems. Although the difference between the two sets of results is of some concern, TP have given cogent argument to support their measurements (TP, 1997; Privalov, private communication). We do not feel we have the expertise to comment on this question and consider only the interpretation of the TP result in what follows.

Counterion Condensation Revisited

We review some of the characteristic properties of the structure of polyelectrolyte solutions: the condensed layer of counterions that forms abruptly at a critical threshold charge density on the polymer chain; the more diffuse Debye-Hückel cloud, which is spatially distinct from the condensed layer; and the entropie release of counterions from the condensed layer as a driving force for the binding of oppositely charged ligands. We present a reminder of the basis of our current understanding in a variety of experiments, simulations, and theories; and we attempt as well to clarify some misunderstandings. We present a new analysis of a lattice model that suggests why the limiting laws for polyelectrolyte thermodynamics have proved to be accurate despite the neglect of polymer-polymer interactions in their original derivation. We sketch recent progress in constructing a potential between counterion and polyion. A counterion located in the interface between condensed layer and Debye cloud is repelled from the polyion, creating a sharp boundary between the two counterion populations.

Thermodynamic properties of a conformationally constrained intramolecular DNA triple helix.

We describe the thermodynamic properties of an intramolecular triple helix with two all-thymine linker loops in which the Hoogsteen strand is covalently crosslinked to the underlying Watson-Crick hairpin duplex by means of a disulfide bridge. We compare these properties to those of the corresponding intramolecular triplex without the disulfide crosslink. Optical and calorimetric measurements reveal that the uncrosslinked parent triplex melts in a biphasic manner above pH 6, with the initial triplex to duplex transition (Hoogsteen strand release) occurring at lower temperatures than subsequent melting of the hairpin helix. By contrast, crosslinking increases the thermal stability of the Hoogsteen transition such that the triplex and underlying hairpin duplex melt as a single transition under all conditions studied. Model independent thermodynamic data obtained by differential scanning calorimetry reveals the crosslink-induced increase in triplex thermal stability corresponds to a free energy stabilization of about 3 kcal/mol, with this stabilization being entirely entropic in origin. In other words, the crosslink is enthalpically neutral, but nevertheless, induces a triplex stabilization of 3 kcal/mol due to a reduction in the entropy change associated with triplex melting. In an effort to define the origin(s) of this entropic impact, we measured the pH and ionic strength dependence of the melting transitions. From a comparison of the melting transitions at different pH values and ionic strengths, we estimate that 0.4 more protons are associated with the crosslinked triplex state than with the uncrosslinked triplex, and 1.3 fewer counterions are released on melting the crosslinked triplex. We discuss how such crosslink-induced changes in proton binding and counterion release, in conjunction with potential changes in hydration and conformational freedom, could combine to give rise to the observed changes in entropy.

Ionic Effects on the Stability and Conformation of Peptide Nucleic Acid Complexes

Peptide nucleic acid (PNA) is a DNA analogue in which the negatively charged sugar phosphate backbone has been substituted by uncharged N-(2-aminoethyl)glycine units. The study of a PNA-DNA duplex and the corresponding DNA-DNA duplex gives a unique opportunity to compare two polyelectrolytes with virtually identical geometry but greatly different linear charge density. The results provide a basis for a study of the applicability of the Poisson-Boltzmann (PB) and counterion condensation (CC) theories. UV and circular dichroism spectroscopy as well as isothermal titration calorimetry (ITC) have been used to study the effect of different ions on the stability and conformation of PNA-DNA, PNA-PNA, and DNA-DNA duplexes having the same base sequences. Cations in general destabilize both antiparallel (N/3') and parallel (N/5') PNA-DNA duplexes whereas they stabilize the DNA-DNA duplex. Studies on the effect of monovalent salt such as NaCl on T-m were carried out over a wide range of salt concentrations (0.01 to 5 M). The decrease in the T-m of the N/3' PNA-DNA duplex with increasing ionic strength in the range of concentrations of 0.01 to 0.5 M, where electrostatic effects predominate, is explained in terms of counterion release upon duplex formation in contrast to the counterion association accompanying the formation of a DNA duplex. The uncharged PNA-PNA duplex shows no significant destabilization in this concentration range. The higher stability of the N/3' PNA-DNA compared to the DNA-DNA duplex (Delta Delta G similar to-7 kcal/mol) is ascribed to more favorable entropic contributions consistent with the counterion release that accompanies the PNA-DNA duplex formation. At high salt concentration (>1 M), where electrostatic contributions saturate, similar trends in the decrease in T-m, were observed for the three types of duplexes irrespective of their backbone charges. The destabilizing effects of a series of Na salts with various monovalent anions on N/3' PNA-DNA and PNA-PNA duplexes were found to follow the Hofmeister series, emphasizing the importance of the hydrophobic interaction between nucleobases for the stability of the PNA complexes in high salt concentration.

Pressure dependence of the helix–coil transition temperature of poly[d(G‐C)

The pressure dependence of the helix-coil transition temperature (Tm) of poly[d(G-C)] was studied as a function of sodium ion concentration in phosphate buffer. The molar volume change of the transition (delta V) was calculated using the Clapeyron equation and calorimetrically determined enthalpies. The delta V of the transition increased from +4.80 (+/- 0.56) to +6.03 (+/- 0.76) mL mol-1 as the sodium ion concentration changed from 0.052 to 1.0M. The van't Hoff enthalpy of the transition calculated from the half-width of the differentiated transition displayed negligible pressure dependence; however, the value of this parameter decreased with increasing sodium ion concentration, indicating a decrease in the size of the cooperative unit. The volume change of the transition exhibits the largest magnitude of any double-stranded DNA polymer measured using this technique. For poly[d(G-C)] the magnitude of the change in delta V with sodium ion concentration (0.94 +/- 0.05 mL mol-1) is approximately one-half that observed for either poly[d(A-T)] or poly(dA).poly(dT). The delta V values are interpreted as arising from changes in the hydration of the polymer due to the release of counterions and changes in the stacking of the bases of the coil form. As a consequence of solvent electrostriction, the release of counterions makes a net negative contribution to the total delta V, implying that disruption of the stacking interactions contributes a positive volume change to the total delta V.(ABSTRACT TRUNCATED AT 250 WORDS)

Salt dependence of calicheamicin-DNA site-specific interactions.

Calicheamicin gamma 1I site-specifically binds and cleaves three closely spaced tetranucleotide sequences embedded in an AT-rich region of a 142 base pair DNA restriction fragment. Cleavage is observed predominantly at the TCCT, TTGT, and ATCT sequences, of which TCCT is the primary cleavage site. The Gibbs free energies required to bind calicheamicin to these sequences within the DNA restriction fragment have been determined as a function of NaCl concentration at pH 8.1 and 0 degrees C and at pH 7.5 and 23 and 0 degrees C. Between 150 mM and 1 M NaCl, calicheamicin binding to all three sequences is insensitive to salt. The insensitivity of calicheamicin binding to salt continues to 50 mM NaCl for the TTGT and ATCT sequences; the delta G values for calicheamicin binding to these sequences are on the order of -7.8 to -7.9 kcal mol-1 over the entire range of NaCl concentrations studied. However, between 150 and 125 mM NaCl, the TCCT sequence displays a sharp transition in the delta G of calicheamicin binding from -7.6 to -8.9 kcal mol-1. Below 125 mM NaCl, the delta G values for calicheamicin binding to the TCCT sequence again are invariant. An analysis of the data in terms of polyelectrolyte theory suggests that counterion release from DNA does not contribute significantly to the energetics of the association and that the association of calicheamicin with specific DNA sequences is dominated by nonionic rather than electrostatic forces. Our results further suggest that some calicheamicin binding/cleavage sites are dependent on flanking sequences.

Modified nucleotides reveal the indirect role of the central base pairs in stabilizing the lac repressor-operator complex.

Guanine residues in the lac operator were replaced by 2-aminopurine or purine analogues, pairing the modified nucleotides with C. The observed equilibrium dissociation constants for lac repressor binding to substituted operators were measured in 10 mM Tris, 150 mM KCl, 0.1 mM EDTA, 0.1 mM DTE, pH 7.6 at 25 degrees C. These measurements revealed five positions that destabilized the complex when substituted with either analogue. Two positions, which are related by a 2-fold symmetry, are in the major groove of the operator thought to directly interact with the protein. Three sites were in the central region of the operator. A purine analogue at a sixth site perturbed the local DNA structure and destabilized the complex. Alkylation interference experiments of the 2-aminopurine substituted operators demonstrated that, of the five affected, two substitutions displayed altered phosphate interference patterns at the phosphate adjacent to the substituted base. For these operators, complex formation was measured in different concentrations of KCl to assess the contribution of counterion release to the bimolecular process. The results indicated that both complexes were similar to wild-type, although minor changes were observed. The Kobs of the complex was then measured when 2-aminopurine or purine analogues were paired with uracil nucleotide, a base pair that serves to stabilize the DNA. The introduction of the new base pairs revealed two effects on the bimolecular interaction. For those operator sites that are thought to perturb the interaction directly, the affinity of the complex was weakened to levels observed for the singly-substituted operators. In contrast, the nucleotides of 2-aminopurine paired with uracil positioned in the central region of the operator served to enhance the stability of the complex. The purine-uracil base pair substitution on the other hand had a significant destabilizing effect on the interaction. We propose that the central base pairs modulate binding of the complex by altering the intrinsic properties of the DNA. Two specific attributes are required to achieve the lowest free energy of interaction. The DNA must have two interstrand hydrogen bonds to stabilize the duplex and it must have properties associated with directional bending or unwinding. This analysis does not rule out contributions by direct interactions between the protein and the central region of the operator but underscores how indirect effects play a major role in complex formation in this system.

Salt Effects on Ligand-DNA Binding: Minor Groove Binding Antibiotics

Salt dependent electrostatic effects play a central role in intermolecular interactions involving nucleic acids. In this paper, the finite-difference solution to the nonlinear Poisson-Boltzmann (NLPB) equation is used to evaluate the salt dependent contribution to the electrostatic binding free energy of the minor groove binding antibiotics DAPI, Hoechst 33258 and netropsin to DNA using detailed molecular structures of the complexes. For each of these systems, a treatment based on the NLPB equation accurately describes the variation of the experimentally observed binding constant with bulk salt concentration. A solvation formalism is developed in which salt effects are described in terms of three free energy contributions: the electrostatic ion-molecule interaction free energy, ΔΔ G o im; the electrostatic ion-ion interaction free energy, ΔΔ G o ii; and the entropic ion organization free energy, ΔΔ G o org. The electrostatic terms, ΔΔ G o im and ΔΔ G o ii, have both enthalpic and entropic components, while the term ΔΔ G o org is purely a cratic entropy. Each of these terms depends significantly on salt dependent changes in the counterion and coion concentrations around the DNA. In each of the systems studied, univalent ions substantially destabilize charged ligand-DNA complexes at physiological salt concentrations. This effect involves a salt dependent redistribution of counterions near the DNA. The free energy associated with the redistribution of counterions upon binding is dominated by the unfavorable change in the electrostatic ion-molecule interactions, ΔΔ G o im, rather than the change in the cratic entropy of ion organization, ΔΔ G o org. In addition, the observed slope of the salt dependence of the free energy is determined by electrostatic ion-molecule and ion-ion interactions as well as the cratic entropy of ion release. These findings are in contrast to models in which the cratic entropy of counterion release drives binding.

Pressure dependence of the melting temperature of dA.dT polymers.

The pressure dependence of the helix-to-coil transition temperature (Tm) of poly[d(A-T)], poly(dA).poly(dT), and poly(dA).poly(dT)2 was studied as a function of sodium chloride concentration. The molar volume change of the transition (delta V) was calculated using the Clapeyron equation and calorimetrically determined enthalpies. The delta V of the transition varied linearly with the logarithm of the salt concentration for both double-stranded polymers. The delta V of poly[d(A-T)] changed from +0.36 to +3.86 cm3 mol-1 as the sodium chloride concentration changed from 0.020 to 1.0 M. For poly(dA).poly-(dT), the delta V varied from +2.60 to +4.59 cm3 mol-1 over the range of 0.020-0.20 M NaCl. No pressure dependence of the van't Hoff enthalpy was observed for the double-helical form of either polymer. The delta V for denaturation of the triple-helical species poly(dA).poly(dT)2 was found to be +7.81 and +10.4 cm3 mol-1 at 1.0 and 3.0 M NaCl, respectively. The observed delta V values are interpreted in terms of changes in the hydration of the polymers arising from release of counterions and changes in the stacking of the bases upon denaturation. Counterion release is assumed to make a net negative contribution to the overall delta V, implying that disruption of the stacking interactions must make a positive volume change to the overall delta V. The difference in the delta V values for the two polymers remains constant as the salt concentration is changed, suggesting a difference in the partial molar volume of their single-stranded forms.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamics of single-stranded RNA binding to oligolysines containing tryptophan.

The equilibrium binding to the synthetic RNA poly(U) of a series of oligolysines containing one, two, or three tryptophans has been examined as a function of pH, monovalent salt concentration (MX), temperature, and Mg2+. Oligopeptides containing lysine (K) and tryptophan (W) of the type KWKp-NH2 and KWKp-CO2 (p = 1-8), as well as peptides containing additional tryptophans or glycines, were studied by monitoring the quenching of the peptide tryptophan fluorescence upon binding poly(U). Equilibrium association constants, K(obs), and the thermodynamic quantities delta G(o)obs, delta H(o)obs, and delta S(o)obs describing peptide-poly(U) binding were measured as well as their dependences on monovalent salt concentration, temperature, and pH. In all cases, K(obs) decreases significantly with increasing monovalent salt concentration, with (delta log K(obs)/delta log [K+]) = -0.74 (+/- 0.04)z, independent of temperature and salt concentration, where z is the net positive charge on the peptide. The origin of these salt effects is entropic, consistent with the release of counterions from the poly(U) upon formation of the complex. Upon extrapolation to 1 M K+, the value of delta G(o)obs is observed to be near zero for all oligolysines binding to poly(U), supporting the conclusion that these complexes are stabilized at lower salt concentrations due to the increase in entropy accompanying the release of monovalent counterions from the poly(U). Only the net peptide charge appears to influence the thermodynamics of these interactions, since no effects of peptide charge distribution were observed. The binding of poly(U) to the monotryptophan peptides displays interesting behavior as a function of the peptide charge. The extent of tryptophan fluorescence quenching, Qmax, is dependent upon the peptide charge for z less than or equal to +4, and the value of Qmax correlates with z-dependent changes in delta H(o)obs and delta S(o)obs(1 M K+), whereas for z greater than or equal to +4, Qmax, delta H(o)obs, and delta S(o)obs (1 M K+) are constant. The correlation between Qmax and delta H(o)obs and delta S(o)obs(1 M K+) suggests a context (peptide charge)-dependence of the interaction of the peptide tryptophan with poly(U). However the interaction of the peptide tryptophan does not contribute substantially to delta G(o)obs for any of the peptides, independent of z, due to enthalpy-entropy compensations. Each of the tryptophans in multiple Trp-containing peptides appear to bind to poly(U) independently, with delta H(o)Trp = -2.9 +/- 0.7, although delta G(o)Trp is near zero due to enthalpy-entropy compensations.(ABSTRACT TRUNCATED AT 400 WORDS)

Calorimetric characterization of parallel-stranded DNA: stability, conformational flexibility, and ion binding

Parallel-stranded DNA is a novel double-stranded, helical form of DNA. Its secondary structure is established by reuerse Watson-Crick base pairing between the bases of the complementary strands, forming two equivalent grooves. A combination of differential scanning calorimetry and temperature-dependent UV spectroscopy techniques have been employed to characterize the stability, conformational flexibility, and counterion binding of two sets of 25-mer deoxyoligonucleotide duplexes containing either exclusively dA.dT base pairs or substitutions with four dG.dC base pairs. These form either parallel-stranded (ps-DleD2 and ps-DSsD6) or antiparallel-stranded (aps-DI.D3 and aps-DSsD7) duplexes. All four duplexes show two-state transition behavior with similar values for the thermodynamic release of counterions, indicating that the charge densities are similar. The parallel duplexes melt with both lower T, values (by 17 and 34 OC, f0.7 C) and lower transition enthalpies (34 and 5 1 kcal-mol-', 13 kcal mol-') than the corresponding antiparallel reference duplexes. These unfavorable differential free energy terms are enthalpically driven, reflecting a reduction in base-stacking interactions and in hydrogen bonding for the case of the duplexes containing dGdC base pairs. Substitution of four dA.dT base pairs of the ps-DIsD2 duplex for four dG.dC base pairs to create the ps-D5.D6 duplex results in a destabilization of 11.9 OC that is entropically driven. The same substitution in the aps duplexes results in a stabilization of 4.9 OC that is enthalpically driven.

The Kinetics of Hydroxyl-Radical-Induced Strand Breakage of Hyaluronic Acid. A Pulse Radiolysis Study Using Conductometry and Laser-Light-Scattering

Hydroxyl radicals were generated radiolytically in N2O- and N2O/O2(4:1)-saturated aqueous solutions of hyaluronic acid. The hydroxyl radicals react rapidly with hyaluronic acid mainly by abstracting carbon-bound H atoms. As a consequence of subsequent free-radical reactions, chain breakage occurs the kinetics of which has been followed using the pulse radiolysis technique. In the absence of oxygen, strand breakage was followed by the change in conductivity induced by the release of cationic counterions condensed at the surface of hyaluronic acid which is a polyanion consisting of subunits of glucuronic acid alternating with N-acetyl-glucosamine. It appears that strand breakage is not due to one single first-order process, however, the contributions of the different components cannot be adequately resolved. At pH 7 the overall half-life is 1.4 ms, in both acid and basic solutions the rate of free-radical induced strand breakage is accelerated (at pH 4.8, t1/2 = 0.6 ms; at pH 10, t1/2 = 0.18 ms). In the absence of oxygen there is no effect of dose rate on the kinetics of strand breakage. In the presence of oxygen in addition to conductometric detection, strand breakage was also followed by changes in low-angle laser light-scattering. These two techniques are complementary in that in this system the conductometry requires high doses per pulse while the light-scattering technique is best operated in the low-dose range. In the presence of oxygen a pronounced dose-rate effect is observed, e.g. at pH 9.7 after a dose of 9.4 Gy the overall half-time is approx. 0.5 s, while after a dose of 6.6 Gy the half-time is approx. 0.23 s. Both the yield and the rate of strand breakage increase with increasing pH, e.g. at pH 7 G(strand breaks) = 0.7 x 10(-7) mol J-1 and at pH 10.4, 4.8 x 10(-7) mol J-7. The radiolytic yields of CO2, H2O2, organic hydroperoxides, O2.- and oxygen consumption have been determined in gamma-irradiated N2O/O2(4:1)-saturated solutions of both hyaluronic acid and beta-cyclodextrin.

Thermodynamic extent of counterion release upon binding oligolysines to single-stranded nucleic acids.

A major contribution to the binding free energy associated with most protein-nucleic acid complexes is the increase in entropy due to counterion release from the nucleic acid that results from electrostatic interactions. To examine this quantitatively, we have measured the thermodynamic extent of counterion release that results from the interaction between single-stranded homopolynucleotides and a series of oligolysines, possessing net charges z = 2-6, 8, and 10. This was accomplished by measuring the salt dependence of the intrinsic equilibrium binding constants--i.e., (delta log Kobs/delta log[K+])--over the range from 6 mM to 0.5 M potassium acetate. These data provide a rigorous test of linear polyelectrolyte theories that have been used to interpret the effects of changes in bulk salt concentration on protein-DNA binding equilibria, since single-stranded nucleic acids have a lower axial charge density than duplex DNA. Upon binding to poly(U), the thermodynamic extent of counterion release per oligolysine charge, z, is 0.71 +/- 0.03, which is significantly less than unity and less than that measured upon binding duplex DNA. These results are most simply interpreted using the limiting law predictions of counterion condensation and cylindrical Poisson-Boltzmann theories, even at the high salt concentrations used in our experiments. Accurate estimates of the thermodynamic extent of counterion binding and release for model systems such as these facilitate our understanding of the energetics of protein-nucleic acid interactions. These data indicate that for simple oligovalent cations, the number of ionic interactions formed in a complex with a linear nucleic acid can be accurately estimated from a measure of the salt dependence of the equilibrium binding constant, if the thermodynamic extent of ion release is known.

Interaction of Escherichia coli translation-initiation factor IF-1 with ribosomes.

The interaction between Escherichia coli translation-initiation factor IF-1 and ribosomes was studied in binding experiments by Airfuge centrifugation. IF-1 binds to the 30S, but not to the 50S, ribosomal subunit and its binding is strongly stimulated by IF-3 and IF-2, either alone or in combination. From the dependence of the Kd of the 30S-subunit--IF-1 complex on ionic strength, it can be concluded that IF-1 binds primarily via an ionic interaction, most likely with the 16S rRNA, with the minimum number of ion pairs involved being 2.7-3.6. The 30S-subunit--IF-1 interaction is unaffected by temperature changes between 11 degrees C and 44 degrees C and is thus accompanied by a negligible enthalpy change. It is concluded that the interaction is an entropy-driven process triggered mainly by the release of counter ions from the RNA phosphates. Titration of 30S-subunit--IF-1 complexes with 50S subunits causes the ejection of the factor indicating that IF-1 is released from the ribosomes during the subunit association step which marks the transition from a 30S-initiation-complex to a 70S initiation complex.

Equilibrium binding of benzo[a]pyrene tetrol to synthetic polynucleotides: sequence selectivity, thermodynamic properties, and ionic strength dependence.

We have investigated the equilibrium binding of racemic 7r,8t,9t,10c-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene to the double-stranded, synthetic polynucleotides poly[d(A-T)], poly[d(G-C)], and poly[d(G-m5C)] at low binding ratios. Difference absorption spectroscopy shows a 10-nm red shift for binding to poly[d(A-T)] and an 11-nm red shift for binding to either poly[d(G-C)] or poly[d(G-m5C)]. The value of delta epsilon for binding is approximately the same for all three hydrocarbon-polynucleotide complexes. Binding of this neutral polycyclic aromatic hydrocarbon derivative to these polynucleotides is dependent upon ionic strength and temperature. Analysis of complex formation employing polyelectrolyte theory shows a greater release of counterions associated with binding to poly[d(A-T)] than with the other two polynucleotides (0.5 and ca. 0.36, respectively). Thus, sequence-selective binding of this hydrocarbon in DNA would be expected to change depending on salt concentration. The temperature dependence of binding was studied at 100 mM Na+ where the equilibrium binding constants for poly[d(A-T)] and poly[d(G-m5C)] are roughly equivalent and 6-fold greater than the binding affinity for poly[d(G-C)]. The binding to poly[d(A-T)] and poly[d(G-C)] is characterized by a delta H omicron = -7.0 kcal/mol, and the large difference in affinity constants arises from differences in negative entropic contributions. Formation of hydrocarbon-poly[d(G-m5C)] complexes is accompanied by a delta H = -9.1 kcal/mol. However, the affinity for poly[d-(G-m5C)] is the same as that for poly[d(A-T)] due to the much more negative entropy associated with binding to poly[d(G-m5C)].

Nonproton ion release by purple membranes exhibits cooperativity as shown by determination of the optical cross-section

The amplitudes of the conductivity transients in photoexcited purple membranes were studied as a function of the energy of the actinic flash to determine the optical cross section of the process giving rise to the conductivity transient. Heating of the solution by the absorbed light causes an additional conductivity change and serves as an internal actinometer; the experiment directly yields the ratio of the cross section of ion release/uptake to that for light absorption. In effect, this counts the number of bacteriorhodopsin (bR) molecules involved in the conductivity transient per photon absorbed. At pH 7 in 0.4-0.5 M NaCl, where the conductivity signals are dominated by nonproton ions, the ratio is between 3 and 4, i.e., excitation of any one of several chromophores generates the same ion release signal. The simplest interpretation is that at pH 7 cooperative conformational changes cause a transient change in the surface charge distribution near all the affected bR molecules, resulting in the transient release of numerous counterions. As a comparison, at pH 4 where the signals are due to protons alone, the cross section data indicate that only a single bR molecule is involved in the proton movements. In this case, the results also show that the sum of the primary forward and reverse quantum yields (for the reactions: bR----K) is 0.88 +/- 0.09.

Molecular mechanical simulations on double intercalation of 9-amino acridine into d(CGCGCGC) X d(GCGCGCG): analysis of the physical basis for the neighbor-exclusion principle.

The neighbor-exclusion principle is one of the most general and interesting rules describing intercalative DNA binding by small molecules. It suggests that such binding can only occur at every other base-pair site, reflecting a very large negative cooperativity in the binding process. We have carried out molecular mechanics and molecular dynamics simulations to study intercalation complexes between 9-amino acridine and the base-paired heptanucleotide d(CGCGCGC) X d(GCGCGCG), in which the neighbor-exclusion principle was both obeyed and violated. Our studies find no stereochemical preference that favors the neighbor-exclusion-obeying structures over the neighbor-exclusion-violating structures. Alternative explanations for the existence of the neighbor-exclusion principle are vibrational entropy effects that we calculate to favor the more flexible neighbor-exclusion models over the more rigid neighbor-exclusion-violating models and polyelectrolyte (counterion release) effects.

Enthalpy and entropy changes for the intercalation of small molecules to DNA. I. Substituted naphthalene monoimides and naphthalene diimides

Calorimetric studies have been performed on the intercalation of a series of nitro and amino substituted naphthalene monoimide cations to calf thymus DNA. For comparison, we also included in the study the unsubstituted naphthalene diimide dication. All of the substituted naphthalene monoimides formed dimers at the concentrations used in the calorimetric titrations, and dimerization constants for these compounds were derived from spectroscopic studies and used in calculating the ΔH parameters from the calorimetric data. The dimerization constants increase in the order 3-NO2MI=4-NO2MI>3-NH2MI>4-NH2MI. For the unsubstituted naphthalene monoimide and 3-NO2MI and 4-NO2MI, the ΔH parameters are within experimental error equal to that found for the naphthalene diimide, i.e., −4.3 kcal-mol−1. Thus, changes in entropy cause the K B for the diimide to be 40 times larger than that for the monoimide. This observation is consistent with the current electrostatic theory for counterion binding to DNA: a dication should cause the release of more counterions than a monocation and produce a more positive ΔS . For the amino substituted naphthalene monoimides, the K B values are similar to the other monoimides, but ΔH =−6.7 kcal-mol−1. We propose that a hydrogen bond is responsible for the unusual enthalpy and entropy effects seen for 3-NH2MI and 4-NH2MI.