Thermodynamics of zinc binding to rhodanese

The binding of zinc ion (Zn²⁺) to rhodanese at two pH values was studied by microcalorimetry and the free energy, enthalpy, and entropy changes determined. Binding exhibited rather large endothermic enthalpy changes quite similar to those observed for zinc-model compound interactions. The large positive entropy changes which accompany binding appear to be a feature common to Zn²⁺-apocarbonic anhydrase systems as well. The correlations between Zn²⁺ interaction with model compounds resembling protein side chains and the thermodynamic values obtained for Zn²⁺-protein interactions suggest that endothermic enthalpies of binding should commonly be observed under slightly acidic to basic conditions. It is found that commercial rhodanese binds Zn²⁺ with moderate to weak affinity by a process that is entropy driven much like that of other Zn²⁺-protein interactions.     

Selective binding of divalent cations toward heme proteins

Potential toxicity of transition metals like Hg, Cu and Cd are well known and their affinity toward proteins is of great concern. This work explores the selective nature of interactions of Cu²⁺, Hg²⁺ and Cd²⁺ with the heme proteins leghemoglobin, myoglobin and cytochrome C. The binding profiles were analyzed using absorbance spectrum and steady-state fluorescence spectroscopy. Thermodynamic parameters like enthalpy, entropy and free energy changes were derived by isothermal calorimetry and consequent binding parameters were compared for these heme proteins. Free energy (DG) values revealed Cu²⁺ binding toward myoglobin and leghemoglobin to be specific and facile in contrast to weak binding for Hg²⁺ or Cd²⁺. Time correlated single photon counting indicated significant alteration in excited state lifetimes for metal complexed myoglobin and leghemoglobin suggesting bimolecular collisions to be involved. Interestingly, none of these cations showed significant affinity for cytochrome c pointing that, presence of conserved sequences or heme group is not the only criteria for cation binding toward heme proteins, but the microenvironment of the residues or a specific folding pattern may be responsible for these differential conjugation profile. Binding of these cations may modulate the conformation and functions of these biologically important proteins.     

Fatty acid and alcohol partitioning with intestinal brush border and erythrocyte membranes

Summary Relative partition coefficients of fatty acids and alcohols between aqueous buffers and biological membranes have been determined from the linear relationship between isotope content of sedimented membranes and aqueous concentration. This technique allows study of highly lipid soluble compounds such as long-chain saturated fatty acids. Rat intestinal brush border membranes and erythrocyte ghost membranes were studied by using homologous series of saturated fatty acids mono-unsaturated fatty acids and 10, 12, and 14 carbon normal alcohols. The influence of chain length on partitioning was similar in the three series with an incremental, free energy of –820 cal/mole per methylene group in brush borders for the saturated fatty acids. Incremental enthalpy and entropy were –1331 cal/mole and –1.64 cal/mole,^oK respectively. Decrease in the partition coefficient due to the double bond (monounsaturated relative to saturated) had an incremental free energy of +1178 cal/mole, incremental enthalpy of –3453 cal/mole, and incremental entropy of –7.34 cal/mole,^oK, while substitution of the hydroxyl for the ionized carboxyl group (pH 7.4) increased the partition coefficient by 72-fold. From these data it must be concluded that the lipid phase of the membrane bilayer is extremely hydrophobic, similar to heptane or polyethylene in polarity.     

Analysis of the bilayer phase transition temperatures of phosphatidylcholines with mixed chains

The analysis of the chain-length dependence of the chain-melting transition temperatures of bilayers composed of lipids with identical chains (Marsh, D. 1991. Biochim. Biophys. Acta. 1062: 1-6) is extended to include lipids with chains of unequal length. The bilayer transition temperatures of saturated asymmetrical phosphatidylcholines are interpreted by assuming that the transition enthalpy and transition entropy are linearly related to the absolute value of the difference in chain length between the sn-1 and sn-2 chains, with constant end contributions. Such an assumption is supported by calorimetric data on phosphatidylcholines of constant mean chainlength and varying chain asymmetry. In particular, a symmetrical linear dependence is observed on the chain asymmetry, Δn, which is centered around a value Δn° that corresponds to the conformational inequivalence of the sn-1 and sn-2 chains. The transition temperature then takes the form: T_t = T_t^∞(n - n_H - h′ | Δn + Δn° |)/(n - n_s - s′ | Δn + Δn°) where n_H, n_s are the end contributions, and h′, s′ are fractional deficits in the incremental transition enthalpy and entropy, respectively, arising from the overlapping regions of the longer chains. Optimization on the transition temperature data for the dependence on chain asymmetry of three series of phosphatidylcholines with constant mean chainlength, n, yields parameters that are capable of predicting the dependence of the transition temperatures on chain asymmetry for other mean chainlengths. The dependence of the transition temperature on mean chainlength for phosphatidylcholines in which the chain asymmetry is maintained constant, as well as the dependence on both mean chain length and chain asymmetry for phosphatidylcholines in which one of the two chains is maintained of constant length, are also described with high accuracy by using the same parameters.     

Elusive affinities

The affinity of two molecules for each other and its temperature dependence are determined by the change in enthalpy, free enthalpy, entropy, and heat capacity upon dissociation. As we know the forces that stabilize-protein–protein or protein–DNA association and the three-dimensional structures of the complex, we can in principle derive values for each one of these parameters. The calculation is done first in gas phase by molecular mechanics, then in solution with the help of hydration parameters calibrated on small molecules. However, estimates of enthalpy and entropy changes in gas phase have excessively large error bars even under the approximation that the components of the complex associate as rigid bodies. No reliable result can be expected at the end. The fit to experimental values derived from binding and calorimetric measurements is poor, except for the dissociation heat capacity. This parameter can be attributed mostly to the hydration step and it correlates with the size of the interface. Many protein–protein complexes have interface areas in the range 1200–2000 Ų and only small conformation changes, so the rigid body approximation applies. It is less generally valid in protein–DNA complexes, which have interfaces covering 2200–3100 Ų, large dissociation heat capacities, and affect both the conformation and the dynamics of their components. © 1995 Wiley-Liss, Inc.     

Studies on the transition of the cristal membrane from the orthodox to the aggregated configuration. III. Loss of coupling ability of adrenal cortex mitochondria in the orthodox configuration

When the cristae of adrenal cortex mitochondria are stabilized in the orthodox configuration by the binding of 20–25 mmoles/mg protein of either Ca²⁺ or free fatty acids (oleic acid), both the capacity for carrying out coupled reactions and the capacity for undergoing energized configurational transitions are lost. The coupled reactions studied included ATP synthesis, divalent cation translocation, monovalent cation trnaslocation, and reversed electron transfer. The coupled processes and energized configurational changes are fully operative when the cristae of adrenal cortex mitochondria are in the aggregated configuration. However, two processes that have been shown to depend on conformational changes (the anaerobic-aerobic proton ejection and energized accumulation of inorganic phosphate) still proceed when mitochondria are in the orthodox configuration. When the mitochondria are initially in the orthodox configuration, addition of divalent cations (Mg²⁺ or Mn²⁺) or albumin induces a transition of the cristae to the aggregated configuration and leads to restoration of all the coupled processes. the orthodox to aggregated transition is reversible and the modulation of this reversibility appears to be one of the key points of control in the mitochondrion and possibly of cellular functions.On leave of absence from the Department of Pathology, Nagoya University School of Medicine, Nagoya, Japan.     

An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins

Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu²⁺, Ni²⁺, Co²⁺, and Zn²⁺). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.     

Synergetic effects of Ca2+ and Cu2+ on phase transition in phosphatidylserine membranes

In complexes of divalent metals with large exchange rate constant (K_H2O) of the coordinated H₂O, such as Ca²⁺ and Cu²⁺, the cubic structure in the ligand field is usually unstable and conformation changes are easily induced. We observed the molecular motion of phosphatidylserine (PS) in an amphipathic solvent (water / methanol / chloroform) by ¹H-NMR and ESR using Ca²⁺ and / or Cu²⁺, which has a similar K_H2O to that of Ca²⁺. We found that Ca²⁺ did not hinder the molecular movements of PS. However, Cu²⁺ reduced the movements of both headgroups and the double bonds in the fatty acids of PS. By addition of both Ca²⁺ and Cu²⁺, phase transition to a soft solid phase in the PS membrane was observed at room temperature. The results indicate that the headgroups are clustered in two-dimensional network with each ligand field displaced from the aqueous phase to the water / oil interface. The structure changes of the polar headgroups after the binding of divalent cations are considered to trigger the phase transition of this acidic phospholipid membrane.     

Thermodynamic Parameters of Opioid Binding in the Presence and Absence of G-Protein Coupling

Abstract

We have investigated the thermodynamic parameters of various opioid ligands interacting with their receptors in rat brain membranes. Affinity constants (K_a), enthalpy and entropy values were obtained from homologous displacement experiments performed at 0, 24 and 33°C. It was found that all the opioid agonists tested ([³H]dihydromorphine (DHM) μ alkaloid; [³H]DAMGO μ peptide; [³H]deltorphin-B δ peptide) display endothermic binding accompanied with a large entropy increase, regardless of their chemical structure (alkaloid or peptide), or of their μ or δ receptor selectivity. In contrast, binding of the antagonist naloxone is exothermic, mainly enthalpy driven. Na⁺ or Mg²⁺ results only in quantitative changes of the thermodynamic parameters. In the presence of the GTP-analog Gpp(NH)p; or Gpp(NH)p + Na⁺; or Gpp(NH)p + Na⁺ + Mg²⁺ the affinity of DHM binding dramatically decreases which might reflect functional uncoupling of the receptor-ligand complex and G-proteins. This altered molecular interactions are also indicated by curvilinear van't Hoff plot and entropy increase. It is concluded that the thermodynamic analysis provides means of determining the underlying driving forces of ligand binding and helps to delineate its mechanism.     

Effect of metal Ions (Ni<Superscript>2+</Superscript>, Cu<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript>) and water coordination on the structure of L-phenylalanine,L-tyrosine,L-tryptophan and their zwitterionic forms

Methods of quantum chemistry have been applied to double-charged complexes involving the transition metals Ni²⁺, Cu²⁺ and Zn²⁺ with the aromatic amino acids (AAA) phenylalanine, tyrosine and tryptophan. The effect of hydration on the relative stability and geometry of the individual species studied has been evaluated within the supermolecule approach. The interaction enthalpies, entropies and Gibbs energies of nine complexes Phe•M, Tyr•M, Trp•M, (M = Ni²⁺, Cu²⁺ and Zn²⁺) were determined at the Becke3LYP density functional level of theory. Of the transition metals studied the bivalent copper cation forms the strongest complexes with AAAs. For Ni²⁺and Cu²⁺ the most stable species are the NO coordinated cations in the AAA metal complexes, Zn²⁺cation prefers a binding to the aromatic part of the AAA (complex II). Some complexes energetically unfavored in the gas-phase are stabilized upon microsolvation.     

Thermodynamics of activation gating in olfactory-type cyclic nucleotide-gated (CNGA2) channels

Olfactory-type cyclic nucleotide-gated (CNG) ion channels open by the binding of cyclic nucleotides to a binding domain in the C-terminus. Employing the Eyring rate theory, we performed a thermodynamic analysis of the activation gating in homotetrameric CNGA2 channels. Lowering the temperature shifted the concentration-response relationship to lower concentrations, resulting in a decrease of both the enthalpy ΔH and entropy ΔS upon channel opening, suggesting that the order of an open CNGA2 channel plus its environment is higher than that of the closed channel. Activation time courses induced by cGMP concentration jumps were used to study thermodynamics of the transition state. The activation enthalpies ΔH^‡ were positive at all cGMP concentrations. In contrast, the activation entropy ΔS^‡ was positive at low cGMP concentrations and became then negative at increasing cGMP concentrations. The enthalpic and entropic parts of the activation energies approximately balance each other at all cGMP concentrations, leaving the free enthalpy of activation in the range between 19 and 21 kcal/mol. We conclude that channel activation proceeds through different pathways at different cGMP concentrations. Compared to the unliganded channel, low cGMP concentrations generate a transitional state of lower order whereas high cGMP concentrations generate a transitional state of higher order.     

The enthalpy of oxidation of flavin mononucleotide

The oxidation enthalpy of reduced flavin mononucleotide at pH 7.0 in 0.2 m phosphate buffer has been studied by determining the heat associated with the reaction: FMNH₂ + 2 Fe(CN)^?3₆ ? FMN + 2 Fe(CN)^?4₆ + 2 H⁺. (a) (The quinone, semiquinone, and hydroquinone forms of FMN are represented as FMN, FMNH, and FMNH₂, respectively.) Calorimetric experiments were performed in a flow microcalorimeter which was modified to prevent sample contamination by oxygen. The enthalpy observed for reaction (a), after correction for dilution and buffer effects, was ?39.2 ± 0.4 kcal (mole FMNH₂)^?1 at 25 °C. The potential difference, ΔE′, developed by reaction (a) was determined potentiometrically and corresponded to a free energy change, ΔG′, of ?30.3 kcal (mole FMNH₂)^?1. The resulting entropy change, ΔS′, was thus calculated to be ?29.8 e.u. Reaction (a) was also studied at temperatures of 7 °C and 35.5 °C. ΔC_p′ for the reaction was calculated as ?155 ± 18 cal deg^?1 (mole FMNH₂)^?1 at 20 °C. ΔH′ for the reaction (b), FMNH₂ ? FMN + H₂, (b) was calculated as +14.2 ± 0.7 kcal mole^?1 at 25 °C, relative to the enthalpy of the hydrogen electrode being identically equal to zero at all values of pH and temperature. The free energy at pH 7.0 for reaction (b), calculated from the potential was found to be ?9.7 kcal mole^?1, which resulted in an entropy for reaction (b) of 80.2 e.u. A thermal titration of reaction (a) was used to calculate the thermodynamic parameters for the formation of semiquinone dimer according to the reaction FMNH₂ + FMN ? (·FMNH)₂. (c) The free energy, enthalpy, and entropy changes for reaction (c) were estimated to be ?6.1 kcal mole^?1, ?7 kcal mole^?1, and ?3 e.u., respectively.     

Titratable transmembrane residues and a hydrophobic plug are essential for manganese import via the Bacillus anthracis ABC transporter MntBC-A

All extant life forms require trace transition metals (e.g., Fe^2/3+, Cu^1/2+, and Mn²⁺) to survive. However, as these are environmentally scarce, organisms have evolved sophisticated metal uptake machineries. In bacteria, high-affinity import of transition metals is predominantly mediated by ABC transporters. During bacterial infection, sequestration of metal by the host further limits the availability of these ions, and accordingly, bacterial ABC transporters (importers) of metals are key virulence determinants. However, the structure–function relationships of these metal transporters have not been fully elucidated. Here, we used metal-sensitivity assays, advanced structural modeling, and enzymatic assays to study the ABC transporter MntBC-A, a virulence determinant of the bacterial human pathogen Bacillus anthracis. We find that despite its broad metal-recognition profile, MntBC-A imports only manganese, whereas zinc can function as a high-affinity inhibitor of MntBC-A. Computational analysis shows that the transmembrane metal permeation pathway is lined with six titratable residues that can coordinate the positively charged metal, and mutagenesis studies show that they are essential for manganese transport. Modeling suggests that access to these titratable residues is blocked by a ladder of hydrophobic residues, and ATP-driven conformational changes open and close this hydrophobic seal to permit metal binding and release. The conservation of this arrangement of titratable and hydrophobic residues among ABC transporters of transition metals suggests a common mechanism. These findings advance our understanding of transmembrane metal recognition and permeation and may aid the design and development of novel antibacterial agents.     

Determination of substrate binding energies in individual subsites of a family 18 chitinase

Thermodynamic parameters for binding of N-acetylglucosamine (GlcNAc) oligomers to a family 18 chitinase, ChiB of Serratia marcescens, have been determined using isothermal titration calorimetry. Binding studies with oligomers of different lengths showed that binding to subsites −2 and +1 is driven by a favorable enthalpy change, while binding to the two other most important subsites, +2 and +3, is driven by entropy with unfavorable enthalpy. These remarkable unfavorable enthalpy changes are most likely due to favorable enzyme-substrate interactions being offset by unfavorable enthalpic effects of the conformational changes that accompany substrate-binding.     

Hormonal control of uterine contraction: Characterization of cyclic AMP-dependent membrane properties in the myometrium

A mitochondria-free membrane fraction prepared from rat myometrium accumulated ⁴⁵Ca²⁺ in the presence of oxalic acid and ATP. The rate of transport of Ca²⁺ into the membranous vesicles was increased by greater than 50% in the presence of 3′,5′-cyclic AMP, but not by 2′,3′-cyclic AMP or 5′-AMP. Membrane ATPase activity was stimulated by cyclic AMP in a manner similar to Ca²⁺-transport. ATPase activity was stimulated by Mg²⁺; slight additional stimulation was obtained in the presence of Na⁺ and K⁺ but not in the presence of Ca²⁺. Despite the cyclic AMP sensitivity of membrane ATPase activity, the absence of any effect of inhibitors of Ca²⁺-transport suggest it has little to do with Ca²⁺ accumulation by the membranes.Cyclic AMP-induced increase in Ca²⁺-transport and membrane ATPase activity was duplicated in vivo by incubating uteri in 10⁻⁴ M isoproterenol prior to membrane isolation. Isoproterenol has been previously shown to increase myometrial cyclic AMP levels, and changes in Ca²⁺-transport by cell membranes in relation to intracellular cyclic AMP levels may be the mechanism through which hormones modulate uterine contractility.     

Thermodynamic analysis of ligand binding and ligand binding-induced tertiary structure formation by the thiamine pyrophosphate riboswitch

The thi-box riboswitch regulates gene expression in response to the intracellular concentration of thiamine pyrophosphate (TPP) in archaea, bacteria, and eukarya. To complement previous biochemical, genetic, and structural studies of this phylogenetically widespread RNA domain, we have characterized its interaction with TPP by isothermal titration calorimetry. This shows that TPP binding is highly dependent on Mg²⁺ concentration. The dissociation constant decreases from ∼200 nM at 0.5 mM Mg²⁺ concentration to ∼9 nM at 2.5 mM Mg²⁺ concentration. Binding is enthalpically driven, but the unfavorable entropy of binding decreases as Mg²⁺ concentration rises, suggesting that divalent cations serve to pre-organize the RNA. Mutagenesis, biochemical analysis, and a new crystal structure of the riboswitch suggest that a critical element that participates in organizing the riboswitch structure is the tertiary interaction formed between the P3 and L5 regions. This tertiary contact is distant from the TPP binding site, but calorimetric analysis reveals that even subtle mutations in L5 can have readily detectable effects on TPP binding. The thermodynamic signatures of these mutations, namely decreased favorable enthalpy of binding and small effects on entropy of binding, are consistent with the P3–L5 association contributing allosterically to TPP-induced compaction of the RNA.     

Perturbations in nucleosome structure from heavy metal association

Heavy metals have the potential to engage in strong bonding interactions and can thus function in essential as well as toxic or therapeutic capacities. We conducted crystallographic analyses of heavy cation binding to the nucleosome core particle and found that Co²⁺ and Ni²⁺ preferentially associate with the DNA major groove, in a sequence- and conformation-dependent manner. Conversely, Rb⁺ and Cs⁺ are found to bind only opportunistically to minor groove elements of the DNA, in particular at narrow AT dinucleotide sites. Furthermore, relative to Mn²⁺ the aggressive coordination of Co²⁺ and Ni²⁺ to guanine bases is observed to induce a shift in histone–DNA register around the nucleosome center by stabilizing DNA stretching over one region accompanied by expulsion of two bases at an opposing location. These ‘softer’ transition metals also associate with multiple histone protein sites, including inter-nucleosomal cross-linking, and display a proclivity for coordination to histidine. Sustained binding and the ability to induce structural perturbations at specific locations in the nucleosome may contribute to genetic and epigenetic mechanisms of carcinogenesis mediated by Co²⁺ and Ni²⁺.     

Binding of transition metals to S100 proteins

The S100 proteins are a unique class of EF-hand Ca²⁺ binding proteins distributed in a cell-specific, tissue-specific, and cell cycle-specific manner in humans and other vertebrates. These proteins are distinguished by their distinctive homodimeric structure, both intracellular and extracellular functions, and the ability to bind transition metals at the dimer interface. Here we summarize current knowledge of S100 protein binding of Zn²⁺, Cu²⁺ and Mn²⁺ ions, focusing on binding affinities, conformational changes that arise from metal binding, and the roles of transition metal binding in S100 protein function.     

Pressure dependence of the helix-coil transition temperature for polynucleic acid helices

Thermal denaturation of DNA's and the corresponding helix–coil transformation of artificial polyribonucleic and polydeoxyribonucleic acids have been studied extensively both theoretically^1–13 and experimentally. ^14–30 Much less work has been carried out on the properties of these polynucleic acids at high pressure, and in particular, on the presure dependence of the helix–coil transition temperature.^31–33 Light-scattering techniques have been used in this study to measure the pressure dependence of the helix–coil transition temperature of the two- and three-stranded helices of polyriboadenylic and polyribouridilic acids and of calf thymus DNA. From the slopes of the transition temperature vs. pressure curves and heats of transition obtained from the literature,^20,34 the following volume changes from these helix–coil transitions have been obtained: (a) ?0.96 cc/mole of nucleotide base pairs for the poly (A + U) transition, (b) +0.35 cc/mole of nucleotide base trios for the poly (A + 2U) transition, and (c) +2.7 cc/mole of nucleotide base pairs for the DNA transition. The relative magnitudes and signs of these volume changes which show that poly (A + U) is destabilized by increased pressure, whereas poly (A + 2U) and calf thymus DNA are stabilized by increased pressure, indicates that further development of the helix–coil transition theory for polynucleotides is needed.