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PubMed2001

Thermodynamics of specific and nonspecific DNA binding by two DNA-binding domains conjugated to fluorescent probes.

The complexes designed in this work combine the sequence-specific binding properties of helix-turn-helix DNA-binding motifs with intercalating cyanine dyes. Thermodynamics of the Hin recombinase and Tc3 transposase DNA-binding domains with and without the conjugated dyes were studied by fluorescence techniques to determine the contributions to specific and nonspecific binding in terms of the polyelectrolyte and hydrophobic effects. The roles of the electrostatic interactions in binding to the cognate and noncognate sequences indicate that nonspecific binding is more sensitive to changes in salt concentration, whereas the change in the heat capacity shows a greater sensitivity to temperature for the sequence-specific complexes in each case. The conjugated dyes affect the Hin DNA-binding domain by acting to anchor a short stretch of amino acids at the N-terminal end into the minor groove. In contrast, the N-terminal end of the Tc3 DNA-binding domain is bound in a well-ordered fashion to the DNA even in the absence of the conjugated dye. The conjugated dye and the DNA-binding domain portions of each conjugate bind noncooperatively to the DNA. The characteristic thermodynamic parameters of specific and nonspecific DNA binding by each of the DNA-binding domains and their respective conjugates are presented.

Amino Acid Sequence↗

PubMed2002

A thermodynamic model for extended complexes of cholesterol and phospholipid.

Studies of monolayer mixtures of certain phospholipids with cholesterol by epifluorescence microscopy and measurement of cholesterol desorption show evidence for the formation of "condensed complexes." A thermodynamic model of these complexes has been developed and has been shown to be generally consistent with observed phase diagrams, cholesterol desorption rates, and electric field susceptibility. Previous work has shown that complexes comprising 10-50 molecules provide good agreement with experimental results. The present study examines the calculated properties of complexes containing very large numbers of molecules and extends the condensed complex model to incorporate the formation of complexes of variable size. Trends in equilibrium composition are similar to those calculated for small complexes. Thermal transitions are continuous, with a strong composition dependence of the breadth of the transition. The average number of molecules in a large complex shows a pronounced dependence on the composition of the reaction mixture. Large complexes have properties of a separate thermodynamic phase.

Biophysical Phenomena↗

PubMed2002

Thermodynamic and kinetic characterization of antisense oligodeoxynucleotide binding to a structured mRNA.

Antisense oligonucleotides act as exogenous inhibitors of gene expression by binding to a complementary sequence on the target mRNA, preventing translation into protein. Antisense technology is being applied successfully as a research tool and as a molecular therapeutic. However, a quantitative understanding of binding energetics between short oligonucleotides and longer mRNA targets is lacking, and selecting a high-affinity antisense oligonucleotide sequence from the many possibilities complementary to a particular RNA is a critical step in designing an effective antisense inhibitor. Here, we report measurements of the thermodynamics and kinetics of hybridization for a number of oligodeoxynucleotides (ODNs) complementary to the rabbit beta-globin (RBG) mRNA using a binding assay that facilitates rapid separation of bound from free species in solution. A wide range of equilibrium dissociation constants were observed, and association rate constants within the measurable range correlated strongly with binding affinity. In addition, a significant correlation was observed of measured binding affinities with binding affinity values predicted using a thermodynamic model involving DNA and RNA unfolding, ODN hybridization, and RNA restructuring to a final free energy minimum. In contrast to the behavior observed for hybridization of short strands, the association rate constant increased with temperature, suggesting that the kinetics of association are related to disrupting the native structure of the target RNA. The rate of cleavage of the RBG mRNA in the presence of ribonuclease H and ODNs of varying association kinetics displayed apparent first-order kinetics, with the rate constant exhibiting binding-limited behavior at low association rates and reaction-limited behavior at higher rates. Implications for the rational design of effective antisense reagents are discussed.

Base Sequence↗

PubMed2002

Folding thermodynamics of model four-strand antiparallel beta-sheet proteins.

The thermodynamic properties for three different types of off-lattice four-strand antiparallel beta-strand protein models interacting via a hybrid Go-type potential have been investigated. Discontinuous molecular dynamic simulations have been performed for different sizes of the bias gap g, an artificial measure of a model protein's preference for its native state. The thermodynamic transition temperatures are obtained by calculating the squared radius of gyration R(g)(2), the root-mean-squared pair separation fluctuation Delta(B), the specific heat C(v), the internal energy of the system E, and the Lindemann disorder parameter Delta(L). Despite these models' simplicity, they exhibit a complex set of protein transitions, consistent with those observed in experimental studies on real proteins. Starting from high temperature, these transitions include a collapse transition, a disordered-to-ordered globule transition, a folding transition, and a liquid-to-solid transition. The high temperature transitions, i.e., the collapse transition and the disordered-to-ordered globule transition, exist for all three beta-strand proteins, although the native-state geometry of the three model proteins is different. However the low temperature transitions, i.e., the folding transition and the liquid-to-solid transition, strongly depend on the native-state geometry of the model proteins and the size of the bias gap.

Biophysical Phenomena↗

PubMed2002

Thermodynamic assessment of the stability of thrombin receptor antagonistic peptides in hydrophobic environments.

In this paper, a general procedure is described to determine thermodynamic parameters associated with the interaction of thrombin receptor antagonistic peptides (TRAPs) with immobilized nonpolar ligands. The results show that these interactions were associated with nonlinear van't Hoff dependencies over a wide temperature range. Moreover, changes in relevant thermodynamic parameters, namely the changes in Gibbs free energy of interaction, DeltaG(0)assoc, enthalpy of interaction, DeltaH(0)assoc, entropy of interaction, DeltaS(0)assoc, and heat capacity, DeltaC(0)p, have been related to the structural properties of these TRAP analogs. The implications of these investigations for the design of thrombin receptor agonists/antagonists with structures stabilized by intramolecular hydrophobic interactions are discussed.

Amino Acid Sequence↗

PubMed2003

Thermodynamics of the hydrophobicity in crystallization of insulin.

For insight into the solvent structure around protein molecules and its role in phase transformations, we investigate the thermodynamics of crystallization of the rhombohedral form of porcine insulin crystals. We determine the temperature dependence of the solubility at varying concentration of the co-solvent acetone, Cac=0%, 5%, 10%, 15%, and 20%, and find that, as a rule, the solubility of insulin increases as temperature increases. The enthalpy of crystallization, undergoes a stepwise shift from approximately -20 kJ mol(-1) at Cac=0%, 5%, and 10% to approximately -55 kJ mol(-1) at Cac=15% and 20%. The entropy change upon crystallization is approximately 35 J mol(-1) K(-1) for the first three acetone concentrations, and drops to approximately -110 J mol(-1) K(-1) at Cac=15% and 20%. DeltaS degrees cryst>0 indicates release of solvent, mostly water, molecules structured around the hydrophobic patches on the insulin molecules' surface in the solution. As Cac increases to 15% and above, unstructured acetone molecules apparently displace the waters and their contribution to DeltaS degrees cryst is minimal. This shifts DeltaS degrees cryst to a negative value close to the value expected for tying up of one insulin molecule from the solution. The accompanying increase in DeltaH degrees cryst suggests that the water structured around the hydrophobic surface moieties has a minimal enthalpy effect, likely due to the small size of these moieties. These findings provide values of the parameters needed to better control insulin crystallization, elucidate the role of organic additives in the crystallization of proteins, and help us to understand the thermodynamics of the hydrophobicity of protein molecules and other large molecules.

Acetone↗

PubMed2004

Modeling electron transfer thermodynamics in protein complexes: interaction between two cytochromes c(3).

Redox protein complexes between type I and type II tetraheme cytochromes c(3) from Desulfovibrio vulgaris Hildenborough are here analyzed using theoretical methodologies. Various complexes were generated using rigid-body docking techniques, and the two lowest energy complexes (1 and 2) were relaxed using molecular dynamics simulations with explicit solvent and subjected to further characterization. Complex 1 corresponds to an interaction between hemes I from both cytochromes c(3). Complex 2 corresponds to an interaction between the heme IV from type I and the heme I from type II cytochrome c(3). Binding free energy calculations using molecular mechanics, Poisson-Boltzmann, and surface accessibility methods show that complex 2 is more stable than complex 1. Thermodynamic calculations on complex 2 show that complex formation induces changes in the reduction potential of both cytochromes c(3), but the changes are larger in the type I cytochrome c(3) (the largest one occurring on heme IV, of approximately 80 mV). These changes are sufficient to invert the global titration curves of both cytochromes, generating directionally in electron transfer from type I to type II cytochrome c(3), a phenomenon of obvious thermodynamic origin and consequences, but also with kinetic implications. The existence of processes like this occurring at complex formation may constitute a natural design of efficient redox chains.

Biophysics↗

PubMed1979

A network thermodynamic two-port element to represent the coupled flow of salt and current. Improved alternative for the equivalent circuit.

A two-port for coupled salt and current flow is created by using the network thermodynamic approach in the same manner as that for coupled solute and volume flow (Mikulecky et al., 1977b; Mikulecky, 1977). This electrochemical two-port has distinct advantages over the equivalent circuit representation and overcomes difficulties pointed out by Finkelstein and Mauro (1963). The electrochemical two-port is used to produce a schematic diagram of the coupled flows through a tissue. The network is superimposable on the tissue morphology and preserves the physical qualities of the flows and forces in each part of an organized structure (e.g., an epithelium). The topological properties are manipulated independently from the constitutive (flow-force) relations. The constitutive relations are chosen from a number of alternatives depending on the detail and rigor desired. With the topology and constitutive parameters specified, the steady-state behavior is simulated with a network simulation program. By using capacitance to represent the filling and depletion of compartments, as well as the traditional electrical capacitances, time-dependent behavior is also simulated. Nonlinear effects arising from the integration of equations describing local behavior (e.g., the Nernst-Planck equations) are dealt with explicitly. The network thermodynamic approach provides a simple, straightforward method for representing a system diagrammatically and then simulating the system's behavior from the diagram with a minimum of mathematical manipulation.

Animals↗

PubMed1980

On the thermodynamics and kinetics of the cooperative binding of bacteriophage T4-coded gene 32 (helix destabilizing) protein to nucleic acid lattices.

In this paper we summarize a series of thermodynamic, and preliminary kinetic, studies on the molecular details and specificity of interaction of phage T4-coded gene 32-protein (GP32) with nucleic acid lattices. It is shown that the binding of GP32 to short (l = 2--8 residues) oligonucleotides is essentially independent of base composition and sugar-type, as well as of salt concentration. In contrast, cooperative (continuous) or isolated binding of GP32 to single-stranded polynucleotides is base and sugar composition-dependent (binding is tighter to DNA than to RNA) and highly dependent on salt concentrations. Binding constants (K), cooperativity parameters (w), and binding site sizes (n) are determined for binding to various nucleic acid lattices under a variety of environmental conditions. These results are used to show that GP32 can bind to nucleic acid lattices in two different conformations, and to characterize the molecular details of these binding species. Further insight into the molecular origins of binding cooperativity is obtained by determining these thermodynamic parameters also for the specifically proteolytically degraded GP32 fragments GP32 I (C-terminal peptide removed) and GP32 III (C- and N-terminal peptides removed). It is also shown that these GP32-nucleic acid binding measurements can be used to provide a quantitative molecular interpretation of the sequential (competitive) binding equilibria involved in the autogenous translational regulation of GP32 synthesis (Lemaire et al., 1978, J. Mol. Biol. 126:73, 1978), and to illustrate some general principles of the development of interactional specificity in cooperatively binding protein-nucleic acid complexes. Preliminary experiments have also been carried out on the kinetics of GP32 association to, and dissociation from, single-stranded nucleic acid lattices. In particular, fluorescence stopped-flow measurements of the dissociation of GP32 from such lattices as a function of lattice saturation (and protein cluster size) can be interpreted to suggest that the protein may translocate ("slide") on the lattice before dissociation, These studies permit an approach to possible rates and mechanisms of such translocation events.

DNA Helicases↗

PubMed1981

Thermodynamics of carbohydrate-lipid interactions.

Thermodynamic analyses of carbohydrate-lipid interactions were performed by investigating the effects of a series of carbohydrates, including monosaccharides, disaccharides, and trisaccharides, on the phase-transition properties of aqueous dispersions of 1,2-dipalmitoyl phosphatidylcholine (DPPC). The temperature of the lipid's main phase transition from the gel to liquid-crystalline phase is essentially unchanged in the presence of carbohydrate. The change in the free energy (delta G) of the transition is zero when a carbohydrate is added to aqueous dispersions of DPPC, while the enthalpy (delta H) and the entropy of the melting of DPPC are decreased. The thermodynamic information was used to examine carbohydrate-lipid interactions. Such interactions were elucidated according to our knowledge of the specific properties of carbohydrates in aqueous solutions and the previously proposed hydrophobic interaction involving hydrocarbon tails of the lipid in aqueous dispersions.

Calorimetry, Differential Scanning↗

PubMed1988

TI-205 nuclear magnetic resonance determination of the thermodynamic parameters for the binding of monovalent cations to gramicidins A and C.

Thermodynamic parameters for the binding of the monovalent cations, Li+, Na+, K+, Rb+, Cs+, NH4+, TI+, and Ag+, to gramicidin A and for the binding of TI+ to gramicidin C, incorporated into lysophosphatidylcholine, have been determined using a combination of TI-205 nuclear magnetic resonance spectroscopy and competition binding. The thermodynamic parameters, enthalpy and entropy, are discussed in terms of a process involving the transfer of cations from an aqueous to amide environment.

Gramicidin↗

PubMed1993

Influence of the local anesthetic tetracaine on the phase behavior and the thermodynamic properties of phospholipid bilayers.

We investigated the influence of the local anesthetic tetracaine on the thermodynamic properties and the temperature- and pressure-dependent phase behavior of the model biomembrane 1,2-dimyristoyl-sn-glycero-3-phosphocholine by using volumetric measurements at temperatures ranging from 0 degrees to 40 degrees C and at pressures from ambient up to 1000 bar. The pVT measurements were complemented by temperature-dependent differential scanning calorimetric measurements. Information about the influence of different concentrations of the local anesthetic on the thermodynamic changes accompanying the lipid phase transitions, and on the thermal expansion coefficient, the isothermal compressibility, and the volume fluctuations of the lipids in their different phases, could be obtained from these experiments. The incorporation of tetracaine leads to an overall disordering of the membrane, as can be inferred from the depression of the main transition temperature and the reduction of the volume change at the main lipid phase transition. The expansion coefficient alpha p and the isothermal compressibility chi T of the lipid bilayer are enhanced by the addition of tetracaine and strongly enhanced values of alpha p and chi T, and the lipid volume fluctuations are found in the direct neighborhood of the main phase transition region. As tetracaine can be viewed as a model system for amphiphilic molecules, these results also provide insight into the general understanding of the physicochemical action of amphiphilic molecules on membranes. The experimental results are compared with recent theoretical predictions for the phase behavior of anesthetic-lipid systems, and the biological relevance of this study is discussed.

Biophysical Phenomena↗

PubMed1994

Conformational transitions of duplex and triplex nucleic acid helices: thermodynamic analysis of effects of salt concentration on stability using preferential interaction coefficients.

For order-disorder transitions of double- and triple-stranded nucleic acid helices, the midpoint temperatures Tm depend strongly on a +/-, the mean ionic activity of uniunivalent salt. Experimental determinations of dTm/d ln a +/- and of the enthalpy change (delta H(o)) accompanying the transition in excess salt permit evaluation of delta gamma, the stoichiometrically weighted combination of preferential interaction coefficients, each of which reflects thermodynamic effects of interactions of salt ions with a reactant or product of the conformational transition (formula; see text) Here delta H(o) is defined per mole of nucleotide by analogy to delta gamma. Application of Eq. 1 to experimental values of delta H(o) and Tm yields values of delta gamma for the denaturation of B-DNA over the range of NaCl concentrations 0.01-0.20 M (Privalov et al. (1969), Biopolymers 8,559) and for each of four order-disorder transitions of poly rA.(poly rU)n, n = 1, 2 over the range of NaCl concentrations 0.01-1.0 M (Krakauer and Sturtevant (1968), Biopolymers 6, 491). For denaturation of duplexes and triplexes, delta gamma is negative and not significantly dependent on a +/-, but delta gamma is positive and dependent on a +/- for the disproportionation transition of poly rA.poly rU duplexes. Quantitative interpretations of these trends and magnitudes of delta gamma in terms of coulombic and excluded volume effects are obtained by fitting separately each of the two sets of thermodynamic data using Eq. 1 with delta gamma PB evaluated from the cylindrically symmetric Poisson-Boltzmann (PB) equation for a standard model of salt-polyelectrolyte solutions. The only structural parameters required by this model are: b, the mean axial distance between the projections of adjacent polyion charges onto the cylindrical axis; and a, the mean distance of closest approach between a salt ion center and the cylindrical axis. Fixing bMS and aMS for the multi-stranded (ordered) conformations, we determined the corresponding best fitted values of bSS and aSS for single-stranded RNA and DNA. The resulting best fitted values of aSS are systematically less than aDS by 2-4 A. Uncertainty in the best-fitted values of bSS is significantly lower than in the aSS, because bMS is known with relatively high precision and because the larger uncertainty in aMS has a relatively small effect on the best-fitted values of bSS:bSS = 3.2 +/- 0.6 A for single-stranded poly rA and poly rU; and bSS = 3.4 +/- 0.2 A for single-stranded DNA. These values are approximately one-halt of those expected for a fully extended single-stranded conformation. With the best fitted values of ass and bss, our calculations of delta gamma PB are in close quantitative agreement with experimental observations on each of five nucleic acid order-disorder transitions.

DNA↗

PubMed1995

Grand canonical Monte Carlo molecular and thermodynamic predictions of ion effects on binding of an oligocation (L8+) to the center of DNA oligomers.

Grand canonical Monte Carlo (GCMC) simulations are reported for aqueous solutions containing excess univalent salt (activities a +/- = 1.76-12.3 mM) and one of the following species: an octacationic rod-like ligand, L8+; a B-DNA oligomer with N phosphate charges (8 < or = N < or = 100); or a complex resulting from the binding of L8+ at the center of an N-mer (24 < or = N < or = 250). Simplified models of these multiply charged species are used in the GCMC simulations to predict the fundamental coulombic contributions to the following experimentally relevant properties: 1) the axial distance over which ligand binding affects local counterion concentrations at the surface of the N-mer; 2) the dependence on N of GCMC preferential interaction coefficients, gamma 32MC identical to delta C3/delta C2l a +/-, T, where C3 and C2 are, respectively, the molar concentrations of salt and the multiply charged species (ligand, N-mer or complex); and 3) the dependence on N of SaKobs identical to d in Kobs/d in a +/- = delta (magnitude of ZJ + 2 gamma 32J), where Kobs is the equilibrium concentration quotient for the binding of L8+ to the center of an N-mer and delta denotes the stoichiometric combination of terms, each of which pertains to a reactant or product J having magnitude of ZJ charges. The participation of electrolyte ions in the ligand binding interaction is quantified by the magnitude of SaKobs, which reflects the net (stoichiometrically weighted) difference in the extent of thermodynamic binding of salt ions to the products and reactants. Results obtained here from GCMC simulations yield a picture of the salient molecular consequences of binding a cationic ligand, as well as thermodynamic predictions whose applicability can be tested experimentally. Formation of the central complex is predicted to cause a dramatic reduction in the surface counterion (e.g., Na+) concentration over a region including but extending well beyond the location of the ligand binding site. For binding a cationic ligand, SaKobs is predicted to be negative, indicating net electrolyte ion release in the binding process. At small enough N, -SaKobs is predicted to decrease strongly toward zero with decreasing N. At intermediate N, -SaKobs appears to exceed its limiting value as N-->infinity.

Cations↗

PubMed1996

Preferential partitioning of melittin into the air/water interface: structural and thermodynamic implications.

The membrane active agent melittin has been investigated with regard to the formation of a Langmuir monolayer and the accordingly induced surface activities. We show that in spite of its considerable solubility in an aqueous medium, this peptide nevertheless largely accumulates in the air/water interface unless the lateral pressure is raised beyond a certain threshold value depending on the pH in the subphase. The true surface concentrations have been determined by means of a recently developed novel method based on thermodynamic principles. It affords an access to the partitioning equilibrium between the surface and subphase domains, provided the latter surrounding is not excessively preferred. In the present case this approach was used to derive quantitative information on the pertinent interfacial structure and thermodynamics. In particular, the apparent molecular area and the Gibbs energy of mutual interaction in the monolayer could be evaluated as a function of the applied surface pressure. The data suggest the existence of two structural conversions in the course of an increasing lateral compression. The surface-associated peptide accordingly assumes three different states of successively reduced area requirements, supposedly owing to an orientational transition involving a straightening up of a helical conformation. This conclusion is corroborated by surface potential measurements reflecting corresponding changes of the effective dipole moment perpendicular to the surface.

Air↗

PubMed1996

Thermodynamics of hydrogen bond and hydrophobic interactions in cyclodextrin complexes.

Values of K, delta G(o), delta H(o), delta S(o) and delta C(po) for the binding reaction of small organic ligands forming 1:1 complexes with either alpha- or beta-cyclodextrin were obtained by titration calorimetry from 15 degrees C to 45 degrees C. A hydrogen bond or hydrophobic interaction was introduced by adding a single functional group to the ligand. The thermodynamics of binding with and without the added group are compared to estimate the contribution of the hydrogen bond or hydrophobic interaction. A change in the environment of a functional group is required to influence the binding thermodynamics, but molecular size-dependent solute-solvent interactions have no effect. For phenolic O-H-O hydrogen bond formation, delta H(o) varies from -2 to -1.4 kcal mol(-1) from 15 degrees C to 45 degrees C, and delta C(p) is increased by 18 cal K(-1) mol(-1). The hydrophobic interaction has an opposite effect: in alpha-cyclodextrin, delta C(po) = -13.3 cal K(-1) mol(-1) per ligand -CH(2)-, identical to values found for the transfer of a -CH(2)-group from water to a nonpolar environment. At room temperature, the hydrogen bond and the -CH(2)-interaction each contribute about -600 cal mol(-1) to the stability (delta G(o)) of the complex. With increased temperature, the hydrogen bond stability decreases (i.e., hydrogen bonds "melt"), but the stability of the hydrophobic interaction remains essentially constant.

Amines↗

PubMed1997

Sequence effects on the relative thermodynamic stabilities of B-Z junction-forming DNA oligomeric duplexes.

Circular dichroism (CD) and ultraviolet absorption techniques were employed in characterizing the sequence-dependent thermodynamic stabilities of B-Z junction-forming DNA duplexes. The Watson strand of the duplexes has the general sequence (5meC-G)4-NXYG-ACTG (where N = A or G and XY represents all permutations of pyrimidine bases). Duplexes were generated by mixing stoichiometric amounts of the complementary strands. Circular dichroism studies indicate that each duplex is fully right-handed at low salt (e.g., 115 mM Na+) but undergoes a salt-induced conformational transition to a structure that possesses both left- and right-handed conformations at high salt (4.5 M Na+), and hence a B-Z junction. Optical melting studies of the DNA duplexes at fixed DNA concentration with total Na+ concentration ranging from 15 mM to 5.0 M were determined. A nonlinear dependence of the melting temperature (Tm) on [Na+] was observed. Thermodynamic parameters at Na+ concentrations of 115 mM and 4.5 M with a wide range of DNA concentrations were determined from UV optical melting studies via construction of van't Hoff plots. A change of a single dinucleotide within these duplexes significantly affected the helix stabilities. The experimentally obtained free energies for the duplex to single-strand transitions were in close agreement with predicted values obtained from two different methods.

Base Sequence↗

PubMed1997

The statistical-thermodynamic basis for computation of binding affinities: a critical review.

Although the statistical thermodynamics of noncovalent binding has been considered in a number of theoretical papers, few methods of computing binding affinities are derived explicitly from this underlying theory. This has contributed to uncertainty and controversy in certain areas. This article therefore reviews and extends the connections of some important computational methods with the underlying statistical thermodynamics. A derivation of the standard free energy of binding forms the basis of this review. This derivation should be useful in formulating novel computational methods for predicting binding affinities. It also permits several important points to be established. For example, it is found that the double-annihilation method of computing binding energy does not yield the standard free energy of binding, but can be modified to yield this quantity. The derivation also makes it possible to define clearly the changes in translational, rotational, configurational, and solvent entropy upon binding. It is argued that molecular mass has a negligible effect upon the standard free energy of binding for biomolecular systems, and that the cratic entropy defined by Gurney is not a useful concept. In addition, the use of continuum models of the solvent in binding calculations is reviewed, and a formalism is presented for incorporating a limited number of solvent molecules explicitly.

Binding Sites↗