Energetics of complex formation of the dna hairpin structure d(GCGAAGC) with aromatic ligands

The energy contributions of various physical interactions to the total Gibbs energy of complex formation of the biologically important DNA hairpin d(GCGAAGC) with aromatic antitumor antibiotics daunomycin and novantrone and mutagens ethidium bromide and proflavin have been calculated. It has been shown that the relatively small value of the total energy of binding of the ligands to the hairpin is the sum of components great in absolute value and different in sign. The contributions of van der Waals interactions and both intra- and intermolecular hydrogen bonds and bonds with aqueous environment have been studied. According to the calculations, the hydrophobic and van der Waals components are energetically favorable in complex formation of the ligands with the hairpin d(GCGAAGC), whereas the electrostatic (with consideration of hydrogen bonds) and entropic components are unfavorable.     

Energetics of complex formation of the DNA hairpin structure d(GCGAAGC) with aromatic ligands

The energy contributions of various physical interactions to the total Gibbs energy of complex formation of the biologically important DNA hairpin d(GCGAAGC) with aromatic antitumor antibiotics daunomycin and novantron and the mutagens ethidium and proflavine have been calculated. It has been shown that the relatively small value of the total energy of binding of the ligands to the hairpin is the sum of components great in absolute value and different in sign. The contributions of van der Waals interactions and both intra- and intermolecular hydrogen bonds and bonds with aqueous environment have been studied. According to the calculations, the hydrophobic and van der Waals components are energetically favorable in complex formation of the ligands with the DNA pairpin d(GCGAAGC), whereas the electrostatic (with consideration of hydrogen bonds) and entropic components are unfavorable.     

Comparative thermodynamic analysis of DNA--protein interactions using surface plasmon resonance and fluorescence correlation spectroscopy

We report a kinetic and thermodynamic analysis of interactions between ssDNA and replication protein A (RPA) using surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) at variable temperature. The two methods yield different values for the Gibbs free energy but nearly the same value for the reaction enthalpy of ssDNA-RPA complex formation. The Gibbs free energy was determined by SPR and FCS to be -62.6 and -54.7 kJ/mol, respectively. The values for the reaction enthalpy are -64.4 and -66.5 kJ/mol. It is concluded that the difference in Gibbs free energy measured by the two methods is due to different reaction entropies. The entropic contribution to the free energy at 25 degrees C is -1.8 kJ/mol for SPR and -11.8 kJ/mol for FCS. In SPR, the reaction is restricted to two dimensions because of immobilization of the DNA molecules to the sensor surface. In contrast, FCS is able to follow complex formation without spatial restrictions. In consequence, the reaction entropy determined from SPR experiments is lower than for FCS experiments.     

Contribution of enthalpy to the energetics of complex formation of aromatic ligands with DNA

The energy contributions of electrostatic, van der Waals interactions, hydrogen bonds, and interactions of charge transfer type to the enthalpy of complex formation of the double-stand DNA with the antitumor antibiotics daunomycin, nogalamycin, and novantron, as well as the mutagens ethidium bromide and proflavine have been calculated. According to the calculations, the van der Waals component (except for nogalamycin) is energetically favorable during complex formation of the antibiotics with DNA, and the contributions of H bonds and electrostatic interactions are unfavorable, with the probability of charge transfer in the complexes being low. It has been shown that the relatively low value of the experimental enthalpy of binding is the sum of components greater in absolute value and different in the sign, which is the cause of large errors in estimating the total enthalpy of complex formation of aromatic ligands with DNA.     

Interpreting protein/DNA interactions: distinguishing specific from non-specific and electrostatic from non-electrostatic components

We discuss the effectiveness of existing methods for understanding the forces driving the formation of specific protein-DNA complexes. Theoretical approaches using the Poisson-Boltzmann (PB) equation to analyse interactions between these highly charged macromolecules to form known structures are contrasted with an empirical approach that analyses the effects of salt on the stability of these complexes and assumes that release of counter-ions associated with the free DNA plays the dominant role in their formation. According to this counter-ion condensation (CC) concept, the salt-dependent part of the Gibbs energy of binding, which is defined as the electrostatic component, is fully entropic and its dependence on the salt concentration represents the number of ionic contacts present in the complex. It is shown that although this electrostatic component provides the majority of the Gibbs energy of complex formation and does not depend on the DNA sequence, the salt-independent part of the Gibbs energy--usually regarded as non-electrostatic--is sequence specific. The CC approach thus has considerable practical value for studying protein/DNA complexes, while practical applications of PB analysis have yet to demonstrate their merit.     

Standard thermodynamic formation properties for the adenosine 5'-triphosphate series.

The criterion for chemical equilibrium at specified temperature, pressure, pH, concentration of free magnesium ion, and ionic strength is the transformed Gibbs energy, which can be calculated from the Gibbs energy. The apparent equilibrium constant (written in terms of the total concentrations of reactants like adenosine 5'-triphosphate, rather than in terms of species) yields the standard transformed Gibbs energy of reaction, and the effect of temperature on the apparent equilibrium constant at specified pressure, pH, concentration of free magnesium ion, and ionic strength yields the standard transformed enthalpy of reaction. From the apparent equilibrium constants and standard transformed enthalpies of reaction that have been measured in the adenosine 5'-triphosphate series and the dissociation constants of the weak acids and magnesium complexes involved, it is possible to calculate standard Gibbs energies of formation and standard enthalpies of formation of the species involved at zero ionic strength. This requires the convention that the standard Gibbs energy of formation and standard enthalpy of formation for adenosine in dilute aqueous solutions be set equal to zero. On the basis of this convention, standard transformed Gibbs energies of formation and standard transformed enthalpies of formation of adenosine 5'-trisphosphate, adenosine 5'-diphosphate, adenosine 5'-monophosphate, and adenosine at 298.15 K, 1 bar, pH = 7, a concentration of free magnesium ions of 10(-3) M, and an ionic strength of 0.25 M have been calculated.     

Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae

Processing bodies (P-bodies) are cytoplasmic RNA granules that contain translationally repressed messenger ribonucleoproteins (mRNPs) and messenger RNA (mRNA) decay factors. The physical interactions that form the individual mRNPs within P-bodies and how those mRNPs assemble into larger P-bodies are unresolved. We identify direct protein interactions that could contribute to the formation of an mRNP complex that consists of core P-body components. Additionally, we demonstrate that the formation of P-bodies that are visible by light microscopy occurs either through Edc3p, which acts as a scaffold and cross-bridging protein, or via the "prionlike" domain in Lsm4p. Analysis of cells defective in P-body formation indicates that the concentration of translationally repressed mRNPs and decay factors into microscopically visible P-bodies is not necessary for basal control of translation repression and mRNA decay. These results suggest a stepwise model for P-body assembly with the initial formation of a core mRNA-protein complex that then aggregates through multiple specific mechanisms.     

On the calculation of Gibbs energy corresponding to enantioselective interactions at a direct HRGC separation of enantiomers

A novel procedure is proposed for the calculation of Gibbs energy corresponding to enantiospecific interactions of 2-(2, 4-dinitrophenoxy)-, 2-phenoxy-, and 2-halogen-n-pentane enantiomers with a beta-cyclodextrin (ChirasilDex) stationary phase under gas chromatographic conditions. This energy is calculated from retention data as a difference between the Gibbs energy of an enantiomer and its corresponding achiral congener. The procedure for the determination of 2-(2,4-dinitrophenoxy)-, 2-phenoxy- and 2-halogen- n-pentane achiral congener retention data is discussed in detail.     

Contribution of changes in translational, rotational, and vibrational degrees of freedom to the energy of complex formation of aromatic ligands with DNA

A method for calculation and analysis of the contribution of changes in translational, rotational, and vibrational degrees of freedom to the energy of complex formation of aromatic compounds with DNA duplex has been developed. The results of calculations of the thermodynamic parameters (ΔG, ΔH, ΔS) indicate that changes in the translational and rotational degrees of freedom destabilize, and changes in the vibrational degree of freedom stabilize the complexes, the energy contribution from the movements under consideration being predominantly of entropic character. It is shown that the energy components of changes in translational, rotational, and vibrational degrees of freedom are in the main comparable with the experimentally determined thermodynamic parameters, which requires consideration of these components in the energy analysis of complex formation of aromatic molecules with DNA. It has been found that the total contribution of changes in translational, rotational, and vibrational degrees of freedom to the Gibbs energy of complexing of aromatic molecules with DNA can be assumed to be on the average the same for different ligands and equal to 8.2 kcal/mol.     

The interaction of blood proteins with brucine

The features of brucine (BC) binding to two blood proteins, bovine hemoglobin (BHb), and bovine serum albumin (BSA), were investigated via fluorescence, circular dichroism and UV/Vis absorption spectroscopy. The results revealed that BC caused the fluorescence quenching of blood proteins by the formation of BC–protein complex. The corresponding thermodynamic parameters were measured at different temperatures. The process of binding BC molecule on protein was a spontaneous molecular interaction procedure in which entropy increased and Gibbs free energy decreased. Hydrophobic and electrostatic interactions play a major role in stabilizing the complex. The molecular docking has been employed to explore the binding site of the BC in BHb and BSA on the Autodock 4.2. The distances r between BC and protein were calculated to be 4.93 and 5.08 nm for BHb, and BSA, respectively. The effect of BC on the conformation of blood proteins was analyzed using CD, synchronous fluorescence and three-dimensional fluorescence spectra.     

Optimization of electrostatic interactions in protein-protein complexes

In this article, we present a statistical analysis of the electrostatic properties of 298 protein-protein complexes and 356 domain-domain structures extracted from the previously developed database of protein complexes (ProtCom, http://www.ces.clemson.edu/compbio/protcom). For each structure in the dataset we calculated the total electrostatic energy of the binding and its two components, Coulombic and reaction field energy. It was found that in a vast majority of the cases (>90%), the total electrostatic component of the binding energy was unfavorable. At the same time, the Coulombic component of the binding energy was found to favor the complex formation while the reaction field component of the binding energy opposed the binding. It was also demonstrated that the components in a wild-type (WT) structure are optimized/anti-optimized with respect to the corresponding distributions, arising from random shuffling of the charged side chains. The degree of this optimization was assessed through the Z-score of WT energy in respect to the random distribution. It was found that the Z-scores of Coulombic interactions peak at a considerably negative value for all 654 cases considered while the Z-score of the reaction field energy varied among different types of complexes. All these findings indicate that the Coulombic interactions within WT protein-protein complexes are optimized to favor the complex formation while the total electrostatic energy predominantly opposes the binding. This observation was used to discriminate WT structures among sets of structural decoys and showed that the electrostatic component of the binding energy is not a good discriminator of the WT; while, Coulombic or reaction field energies perform better depending upon the decoy set used.     

Aliphatics vs. aromatics hydration thermodynamics

By comparing the hydration thermodynamics of benzene with that of a hypothetical aliphatic hydrocarbon having the same accessible surface area (ASA) of benzene, Makhatadze and Privalov concluded that the whole difference is due to the weak H-bonds that water forms with the aromatic ring. The formation of such H-bonds would be characterized by a negative Gibbs energy change, slightly increasing in magnitude with temperature, and a positive entropy change over a large temperature range. The latter thermodynamic feature is not physically reliable for the formation of H-bonds. In the present article, by using a statistical mechanical dissection scheme of hydration, a microscopic interpretation for the numbers obtained by Makhatadze and Privalov is proposed. The difference in hydration Gibbs energy should be attributed to the different strength of van der Waals interactions that benzene can do with water, owing to the larger polarizability of the aromatic ring with respect to an aliphatic hydrocarbon of equal size. In addition, the difference in hydration entropy should account for the different extent of H-bond reorganization upon the insertion of benzene and the corresponding aliphatic hydrocarbon in water.     

Gibbs free energy of transfer of glycine and diglycine from water to alkylurea solutions. Measured vs. calculated values

Ternary systems comprising water (1), glycine (diglycine) (2) and alkylurea (3) have been investigated using vapor pressure osmometry. Equations were obtained in terms of the molalities of the solutes for the activity coefficients of glycine and diglycine in these systems. The alkylureas used were methyl-, ethyl- and N, N'-dimethylurea. Using the activity coefficients the Gibbs free energy of transfer at infinite dilution of component 2 from water to alkylurea solutions was determined. Since the enthalpies of transfer are known, the corresponding entropies could also be obtained. Calculation of the Gibbs free energy of transfer at infinite dilution of component 2 rests on the assumption that it can be divided into two parts: the difference between the Gibbs free energy of cavity formation and that of interaction in the alkylurea solution and water, respectively. The first part was calculated by scaled particle theory using experimental density and surface tension data. The second part was taken to be due mainly to the change in dipole-dipole interactions.     

Assessment by sedimentation equilibrium analysis of a heterologous macromolecular interaction in the presence of self-association: interaction of S5 with S8

The proteins S5 and S8 from the Escherichia coli 30S ribosomal subunit have been examined by sedimentation equilibrium methods for behavior in solution as isolated components and in mixtures. The means of resolving two simultaneous associations in this system is discussed, and the energy of association of S5 and S8 is reported. It was found that protein S5 from the MRE 600 strain tends to self-associate weakly at 4 degree C in a manner that can be described as an isodesmic self-association with an association constant and corresponding standard Gibbs free energy equal to (7.7 +/- 0.7) X 10(3) M-1 and -4.9 +/- 0.1 kcal/mol, respectively. Protein S8 was found to have a molecular weight of 15800 and was monomeric in a pure state. Mixtures of S5 and S8 clearly demonstrated the presence of an S5-S8 complex in addition to the self-association of S5. The equilibrium constant of association for the formation of a simple S5-S8 complex at 4 degree C and the corresponding standard Gibbs free energy were found to be (5.5 +/- 1.0) X 10(4) M-1 and -6.0 +/- 0.1 kcal/mol, respectively.     

Studies by means of 1H NMR spectroscopy of complex formation of aromatic biologically active compounds with antibiotic topotecan

The analysis of heteroassociation of antibiotic topotecan (TPT) with aromatic biologically active compounds (BAC): caffeine, mutagens ethidium bromide and proflavine, antibiotic daunomycin, vitamins flavin-mononucleotide and nicotinamide, has been carried out in the work using 1H NMR spectroscopy data. The equilibrium constants of heteroassociation and induced chemical shifts of the protons have been obtained in the complexes with BAC. It is found that the complex formation TPT-BAC has the nature of stacking of the chromophores, additionally stabilized in the case of proflavine by intermolecular hydrogen bond. Calculation of the basic components of the Gibbs free energy of the complexation reactions is carried out, and the factors which stabilize and destabilize the heterocomplexes of molecules are revealed.     

On the calculation of binding free energies using continuum methods: Application to MHC class I protein-peptide interactions

This paper describes a methodology to calculate the binding free energy (ΔG) of a protein-ligand complex using a continuum model of the solvent. A formal thermodynamic cycle is used to decompose the binding free energy into electrostatic and non-electrostatic contributions. In this cycle, the reactants are discharged in water, associated as purely nonpolar entities, and the final complex is then recharged. The total electrostatic free energies of the protein, the ligand, and the complex in water are calculated with the finite difference Poisson-Boltzmann (FDPB) method. The nonpolar (hydrophobic) binding free energy is calculated using a free energy-surface area relationship, with a single alkane/water surface tension coefficient (γ_aw). The loss in backbone and side-chain configurational entropy upon binding is estimated and added to the electrostatic and the nonpolar components of ΔG. The methodology is applied to the binding of the murine MHC class I protein H-2K^b with three distinct peptides, and to the human MHC class I protein HLA-A2 in complex with five different peptides. Despite significant differences in the amino acid sequences of the different peptides, the experimental binding free energy differences (ΔΔG_exp) are quite small (<0.3 and <2.7 kcal/mol for the H-2K^b and HLA-A2 complexes, respectively). For each protein, the calculations are successful in reproducing a fairly small range of values for ΔΔG_calc (<4.4 and <5.2 kcal/mol, respectively) although the relative peptide binding affinities of H-2K^b and HLA-A2 are not reproduced. For all protein-peptide complexes that were treated, it was found that electrostatic interactions oppose binding whereas nonpolar interactions drive complex formation. The two types of interactions appear to be correlated in that larger nonpolar contributions to binding are generally opposed by increased electrostatic contributions favoring dissociation. The factors that drive the binding of peptides to MHC proteins are discussed in light of our results.     

Studies of the interaction between paraquat and bovine hemoglobin

The interaction between paraquat (PQ) and bovine hemoglobin (BHb) was investigated using fluorescence and UV/vis absorption spectroscopy. The reactivity of the heme centers with superoxide anions formed by PQ was judged on the basis of the decrease of the Soret band. The experimental results showed that the fluorescence quenching of BHb by PQ was a result of the formation of PQ-BHb complex; static quenching was confirmed to result in the fluorescence quenching. The binding site number n, apparent binding constant K(A) and corresponding thermodynamic parameters were measured at different temperatures. The process of binding PQ molecule on BHb was a spontaneous molecular interaction procedure in which entropy increased and Gibbs free energy decreased. Hydrophobic and electrostatic interactions played a major role in stabilizing the complex. The effect of PQ on the conformation of BHb was analyzed using synchronous fluorescence spectroscopy.     

Monte Carlo simulation of lipid mixtures: finding phase separation.

The nonideal mixing of phosphatidylserine (PS) and phosphatidylcholine (PC) binary lipid mixtures was studied by computer simulation based on a model wherein the excess energy of mixing is divided between an electrostatic term and one adjustable term delta Em that includes all other nonideal interactions. The lateral distribution of the lipids and the energy of the mixtures were obtained by using Kawasaki relaxation in a canonical ensemble. The Gibbs free energies were calculated by Kirkwood's coupling parameter method. The simulation results are strongly dependent on simulation size for sizes smaller than about 1000 lipids. Nonideal interaction between lipids can result in large scale separation of lipid phases of different composition at reasonable delta Em values as well as clustering of like lipids. In plots of total Gibbs free energy of mixing versus PS mole fraction in PS/PC, the boundaries of the two phase region could be accurately determined. The electrostatic interaction influences cluster size and shape, and also the composition of phases in the two-phase region.     

Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability

Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor-mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.     

Elucidating protein thermodynamics from the three-dimensional structure of the native state using network rigidity

Given the three-dimensional structure of a protein, its thermodynamic properties are calculated using a recently introduced distance constraint model (DCM) within a mean-field treatment. The DCM is constructed from a free energy decomposition that partitions microscopic interactions into a variety of constraint types, i.e., covalent bonds, salt-bridges, hydrogen-bonds, and torsional-forces, each associated with an enthalpy and entropy contribution. A Gibbs ensemble of accessible microstates is defined by a set of topologically distinct mechanical frameworks generated by perturbing away from the native constraint topology. The total enthalpy of a given framework is calculated as a linear sum of enthalpy components over all constraints present. Total entropy is generally a nonadditive property of free energy decompositions. Here, we calculate total entropy as a linear sum of entropy components over a set of independent constraints determined by a graph algorithm that builds up a mechanical framework one constraint at a time, placing constraints with lower entropy before those with greater entropy. This procedure provides a natural mechanism for enthalpy-entropy compensation. A minimal DCM with five phenomenological parameters is found to capture the essential physics relating thermodynamic response to network rigidity. Moreover, two parameters are fixed by simultaneously fitting to heat capacity curves for histidine binding protein and ubiquitin at five different pH conditions. The three free parameter DCM provides a quantitative characterization of conformational flexibility consistent with thermodynamic stability. It is found that native hydrogen bond topology provides a key signature in governing molecular cooperativity and the folding-unfolding transition.