DNA-binding cytotoxic alkaloids: comparative study of the energetics of binding of berberine, palmatine, and coralyne

Deoxyribonucleic acid is the site of storage and retrieval of genetic information through interaction with proteins and other small molecules. In the present study, the interaction of two natural cytotoxic protoberberine plant alkaloids, berberine and palmatine, and a synthetic derivative, coralyne, with mammalian herring testis DNA was investigated using a combination of isothermal titration calorimetry, differential scanning calorimetry, and optical melting experiments to characterize the energetics of their binding. The binding constants of these alkaloids to DNA under identical conditions were evaluated from the UV melting data, and the enthalpy of binding was elucidated from isothermal titration studies. The binding constants of berberine, palmatine, and coralyne to DNA were found to be 1.15 x 10(4), 2.84 x 10(4), and 3.5 x 10(6) M(-1) at 20 degrees C in buffer of 20 mM [Na+]. Parsing of the free energy change of the interaction observed into polyelectrolytic and nonpolyelectrolytic components suggested that although these alkaloids are charged, the major contributor of about 75% of the binding free energy arises from the nonpolyelectrolytic forces. The binding in case of palmatine and coralyne was predominantly enthalpy driven with favoring smaller entropy terms, while that of berberine was favored by both negative enthalpy and positive entropy changes. Temperature dependence of the binding enthalpies determined from ITC studies in the range 20-40 degrees C was used to calculate the binding-induced change in heat capacity (DeltaC(o)(p)) values as -117, -135, and -157 cal/mol K, respectively, for berberine, palmatine, and coralyne. Taken together, the results suggest that the DNA binding of the planar synthetic coralyne is stronger and thermodynamically more favored compared to the buckled natural berberine and palmatine.     

Stability and binding properties of wild-type and c17s mutated human sterol carrier protein 2.

The temperature- and solvent-induced denaturation of both the SCP2 wild-type and the mutated protein c71s were studied by CD measurements at 222 nm. The temperature-induced transition curves were deconvoluted according to a two-state mechanism resulting in a transition temperature of 70.5 degrees C and 59.9 degrees C for the wild-type and the c71s, respectively, with corresponding values of the van't Hoff enthalpies of 183 and 164 kJ/mol. Stability parameters characterizing the guanidine hydrochloride denaturation curves were also calculated on the basis of a two-state transition. The transitions of the wild-type occurs at 0.82 M GdnHCl and that of the c71s mutant at 0.55 M GdnHCl. These differences in the half denaturation concentration of GdnHCl reflect already the significant stability differences between the two proteins. A quantitative measure are the Gibbs energies DeltaG(0)(D)(buffer) at 25 degrees C of 15.5 kJ/mol for the wild-type and 8.0 kJ/mol for the mutant. We characterized also the alkyl chain binding properties of the two proteins by measuring the interaction parameters for the complex formation with 1-O-Decanyl-beta-D-glucoside using isothermal titration microcalorimetry. The dissociation constants, K(d), for wild-type SCP2 are 335 microM at 25 degrees C and 1.3 mM at 35 degrees C. The corresponding binding enthalpies, DeltaH(b), are -21. 5 kJ/mol at 25 degrees C and 72.2 kJ/mol at 35 degrees C. The parameters for the c71s mutant at 25 degrees C are K(d)=413 microM and DeltaH(b)=16.6 kJ/mol. These results suggest that both SCP2 wild-type and the c71s mutant bind the hydrophobic compound with moderate affinity.     

Effects of monovalent anions on a temperature-dependent heat capacity change for Escherichia coli SSB tetramer binding to single-stranded DNA

We have previously shown that the linkage of temperature-dependent protonation and DNA base unstacking equilibria contribute significantly to both the negative enthalpy change (DeltaH(obs)) and the negative heat capacity change (DeltaC(p,obs)) for Escherichia coli SSB homotetramer binding to single-stranded (ss) DNA. Using isothermal titration calorimetry we have now examined DeltaH(obs) over a much wider temperature range (5-60 degrees C) and as a function of monovalent salt concentration and type for SSB binding to (dT)(70) under solution conditions that favor the fully wrapped (SSB)(65) complex (monovalent salt concentration >or=0.20 M). Over this wider temperature range we observe a strongly temperature-dependent DeltaC(p,obs). The DeltaH(obs) decreases as temperature increases from 5 to 35 degrees C (DeltaC(p,obs) <0) but then increases at higher temperatures up to 60 degrees C (DeltaC(p,obs) >0). Both salt concentration and anion type have large effects on DeltaH(obs) and DeltaC(p,obs). These observations can be explained by a model in which SSB protein can undergo a temperature- and salt-dependent conformational transition (below 35 degrees C), the midpoint of which shifts to higher temperature (above 35 degrees C) for SSB bound to ssDNA. Anions bind weakly to free SSB, with the preference Br(-) > Cl(-) > F(-), and these anions are then released upon binding ssDNA, affecting both DeltaH(obs) and DeltaC(p,obs). We conclude that the experimentally measured values of DeltaC(p,obs) for SSB binding to ssDNA cannot be explained solely on the basis of changes in accessible surface area (ASA) upon complex formation but rather result from a series of temperature-dependent equilibria (ion binding, protonation, and protein conformational changes) that are coupled to the SSB-ssDNA binding equilibrium. This is also likely true for many other protein-nucleic acid interactions.     

A calorimetric study of the binding of S-alkylglutathiones to glutathione S-transferase.

The binding of three competitive glutathione analogue inhibitors (S-alkylglutathione derivatives) to glutathione S-transferase from Schistosoma japonicum, SjGST, has been investigated by isothermal titration microcalorimetry at pH 6.5 over a temperature range of 15--30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that no protons are exchanged during the binding of S-alkylglutathione derivatives. Thus, at pH 6.5, the protons released during the binding of substrate may be from its thiol group. Calorimetric analyses show that S-methyl-, S-butyl-, and S-octylglutathione bind to two equal and independent sites in the dimer of SjGST. The affinity of these inhibitors to SjGST is greater as the number of methylene groups in the hydrocarbon side chain increases. In all cases studied, Delta G(0) remains invariant as a function of temperature, while Delta H(b) and Delta S(0) both decrease as the temperature increases. The binding of three S-alkylglutathione derivatives to the enzyme is enthalpically favourable at all temperatures studied. The temperature dependence of the enthalpy change yields negative heat capacity changes, which become less negative as the length of the side chain increases.     

A product inhibition study on adenosine deaminase by spectroscopy and calorimetry

Kinetic and thermodynamic studies have been made on the effect of the inosine product on the activity of adenosine deaminase in a 50 mM sodium phosphate buffer, pH 7.5, at 27 degrees C using UV spectrophotometry and isothermal titration calorimetry (ITC). A competitive inhibition was observed for inosine as a product of the enzymatic reaction. A graphical-fitting method was used for determination of the binding constant and enthalpy of inhibitor binding by using isothermal titration microcalorimetry data. The dissociation-binding constant is equal to 140 microM by the microcalorimetry method, which agrees well with the value of 143 microM for the inhibition constant that was obtained from the spectroscopy method     

Binding of berberine to human telomeric quadruplex - spectroscopic, calorimetric and molecular modeling studies

This study examines the characteristics of binding of berberine to the human telomeric d[AG(3)(T(2)AG(3))(3)] quadruplex. By employing UV-visible spectroscopy, fluorescence spectroscopy and isothermal titration calorimetry, we found that the binding affinity of berberine to the human telomeric quadruplex is 10(6). The complete thermodynamic profile for berberine binding to the quadruplex, at 25 degrees C, shows a small negative enthalpy (DeltaH) of -1.7 kcal.mol(-1), an entropy change with TDeltaS of +6.5 kcal.mol(-1), and an overall favorable free energy (DeltaG) of -8.2 kcal.mol(-1) .Through the temperature dependence of DeltaH, we obtained a heat capacity (DeltaC(p)) of -94 (+/- 5) cal.mol(-1).K(-1). The osmotic stress method revealed that there is an uptake of 13 water molecules in the complex relative to the free reactants. Furthermore, the molecular modeling studies on different quadruplex-berberine complexes show that berberine stacking at the external G-quartet is mainly aided by the pi-pi interaction and the stabilization of the high negative charge density of O6 of guanines by the positively charged N7 of berberine. The theoretical heat capacity (DeltaC(p)) values for quadruplex-berberine models are -89 and -156 cal.mol(-1).K(-1).     

Thermodynamics and folding pathway of tetraloop receptor-mediated RNA helical packing

Little is known about the thermodynamic forces that drive the folding pathways of higher-order RNA structure. In this study, we employ calorimetric [isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC)] and spectroscopic (NMR and UV) methods to characterize the thermodynamics of the GAAA tetraloop-receptor interaction, utilizing a previously described bivalent construct. ITC studies indicate that the bivalent interaction is enthalpy driven and highly stable, with a binding constant (K(obs)) of 5.5x10(6) M(-1) and enthalpy (DeltaH(obs)(o)) of -33.8 kcal/mol at 45 degrees C in 20 mM KCl and 2 mM MgCl(2). Thus, we derive the DeltaH(obs)(o) for a single tetraloop-receptor interaction to be -16.9 kcal/mol at these conditions. UV absorbance data indicate that an increase in base stacking quality contributes to the enthalpy of complex formation. These highly favorable thermodynamics are consistent with the known critical role for the tetraloop-receptor motif in the folding of large RNAs. Additionally, a significant heat capacity change (DeltaC(p,obs)(o)) of -0.24 kcal mol(-1) K(-1) was determined by ITC. DSC and UV-monitored thermal denaturation experiments indicate that the bivalent tetraloop-receptor construct follows a minimally five-state unfolding pathway and suggest the observed DeltaC(p,obs)(o) for the interaction results from a temperature-dependent unbound receptor RNA structure.     

Energetics of solvent and ligand-induced conformational changes in alpha-lactalbumin

The energetics of structural changes in the holo and apo forms of a-lactalbumin and the transition between their native and denatured states induced by binding Ca2+ and Na+ have been studied by differential scanning and isothermal titration microcalorimetry and circular dichroism spectroscopy under various solvent conditions. Removal of Ca2+ from the protein enhances its sensitivity to pH and ionic conditions due to noncompensated negative charge-charge interactions at the cation binding site, which significantly reduces its overall stability. At neutral pH and low ionic strength, the native structure of apo-alpha-lactalbumin is stable below 14 C and undergoes a conformational change to a native-like molten globule intermediate at temperatures above 25 degrees C. The denaturation of either holo- or apo-alpha-lactalbumin is a highly cooperative process that is characterized by an enthalpy of similar magnitude when calculated at the same temperature. Measured by direct calorimetric titration, the enthalpy of Ca2+-binding to apo-LA at pH 7.5 is -7.1 kJ mol(-1) at 5.0 degrees C. which is essentially invariant to protonation effects. This small enthalpy effect infers that stabilization of alpha-lactalbumin by Ca2+ is primarily an entropy driven process, presumably arising from electrostatic interactions and the hydration effect. In contrast to the binding of calcium, the interaction of sodium with apo-LA does not produce a noticeable heat effect and is characterized by its ionic nature rather than specific binding to the metal-binding site. Characterization of the conformational stability and ligand binding energetics of alpha-lactalbumin as a function of solvent conditions furnishes significant insight regarding the molecular flexibility and regulatory mechanism mediated by this protein.     

Thermodynamics of the binding of AMP to glycogen phosphorylase a

The binding of AMP to rabbit muscle glycogen phosphorylase a (EC 2.4.1.1.) has been studied by equilibrium dialysis and isothermal microcalorimetry at pH 6.9 over a temperature range of 25 degrees C to 35 degrees C. Thermal titration experiments were carried out in various buffer systems. We have found by these methods that a certain number of protons are released when the protein binds to the ligand and are taken up by the buffer. The tetramer of phosphorylase a has been shown to have four equal and independent, non-cooperative binding sites for AMP at 25 degrees C, 30 degrees C, and 35 degrees C; these sites can be assigned to the so-called nucleotide or, activator, sites in the protein. The binding constants together with the changes in Gibbs energy, enthalpy, and entropy per site for the AMP binding were calculated at each temperature. A negative delta Cp value of -2.3 +/- 0.2 J K-1 (AMP bound)-1 was obtained for this binding process. The hydrophobic and vibrational contributions of the heat capacity and entropy changes have been resolved by the method described by Sturtevant (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240). From this analysis, it appears that the binding is, in all cases, enthalpy-driven, the two entropic contributions, hydrophobic and vibrational, having opposing effects.     

Evidence for a C4b binding site on the C2b domain of C2

We raised murine mAb against human C protein C2. The representative mAb 3A3.3 (IgG1 kappa) recognized an epitope on the C2b domain of C2, as determined by binding and inhibition of binding radioassays. The hemolytic activity of purified human C2 and of C2 in normal human serum was inhibited by the mAb. The rate of decay of the C3-convertase at 30 degrees C was not affected by the mAb. C2 binding to EAC4b was inhibited by intact IgG and the Fab fragment of the mAb; 50% inhibition required 1 microgram/ml of either. The data suggest the presence of a C4b-binding site on the C2b domain of C2 and that the mAb recognizes an epitope at, or adjacent to, this site. The C2b portion of the C2 molecule may be important in assembly of the classical pathway C3-convertase.     

Global effects of the energetics of coenzyme binding: NADPH controls the protein interaction properties of human cytochrome P450 reductase

The thermodynamics of coenzyme binding to human cytochrome P450 reductase (CPR) and its isolated FAD-binding domain have been studied by isothermal titration calorimetry. Binding of 2',5'-ADP, NADP(+), and H(4)NADP, an isosteric NADPH analogue, is described in terms of the dissociation binding constant (K(d)), the enthalpy (DeltaH(B)) and entropy (TDeltaS(B)) of binding, and the heat capacity change (DeltaC(p)). This systematic approach allowed the effect of coenzyme redox state on binding to CPR to be determined. The recognition and stability of the coenzyme-CPR complex are largely determined by interaction with the adenosine moiety (K(d2)(')(,5)(')(-ADP) = 76 nM), regardless of the redox state of the nicotinamide moiety. Similar heat capacity change (DeltaC(p)) values for 2',5'-ADP (-210 cal mol(-)(1) K(-)(1)), NADP(+) (-230 cal mol(-)(1) K(-)(1)), and H(4)NADP (-220 cal mol(-)(1) K(-)(1)) indicate no significant contribution from the nicotinamide moiety to the binding interaction surface. The coenzyme binding stoichiometry to CPR is 1:1. This result validates a recently proposed one-site kinetic model [Daff, S. (2004) Biochemistry 43, 3929-3932] as opposed to a two-site model previously suggested by us [Gutierrez, A., Lian, L.-Y., Wolf, C. R., Scrutton, N. S., and Roberts, C. G. K. (2001) Biochemistry 40, 1964-1975]. Calorimetric studies in which binding of 2',5'-ADP to CPR (TDeltaS(B) = -13400 +/- 200 cal mol(-)(1), 35 degrees C) was compared with binding of the same ligand to the isolated FAD-binding domain (TDeltaS(B) = -11200 +/- 300 cal mol(-)(1), 35 degrees C) indicate that the number of accessible conformational substates of the protein increases upon 2',5'-ADP binding in the presence of the FMN-binding domain. This pattern was consistently observed along the temperature range that was studied (5-35 degrees C). This contribution of coenzyme binding energy to domain dynamics in CPR agrees with conclusions from previous temperature-jump studies [Gutierrez, A., Paine, M., Wolf, C. R., Scrutton, N. S., and Roberts, G. C. K. (2002) Biochemistry 41, 4626-4637]. A combination of calorimetry and stopped-flow spectrophotometry kinetics experiments showed that this linkage between coenzyme binding energetics and diffusional domain motion impinges directly on the molecular recognition of cytochrome c by CPR. Single-turnover reduction of cytochrome c by CPR (k(max) = 15 s(-)(1), K(d) = 37 microM) is critically coupled to coenzyme binding through ligand-induced motions that enable the FMN-binding domain to overcome a kinetically unproductive conformation. This is remarkable since the FMN-binding domain is not directly involved in coenzyme binding, the NADP(H) binding site being fully contained in the FAD-binding domain. Sequential rapid mixing measurements indicate that harnessing of coenzyme binding energy to the formation of a kinetically productive CPR-cytochrome c complex is a highly synchronized event. The inferred half-time for the decay of this productive conformation (tau(50)) is 330 +/- 70 ms only. Previously proposed structural and kinetic models are discussed in light of these findings.     

Thermodynamics of the folding of D-glyceraldehyde-3-phosphate dehydrogenase assisted by protein disulfide isomerase studied by microcalorimetry.

Thermodynamics of the refolding of denatured D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) assisted by protein disulfide isomerase (PDI), a molecular chaperone, has been studied by isothermal microcalorimetry at different molar ratios of PDI/GAPDH and temperatures using two thermodynamic models proposed for chaperone-substrate binding and chaperone-assisted substrate folding, respectively. The binding of GAPDH folding intermediates to PDI is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong enthalpy-entropy compensation and weak temperature dependence of Gibbs free energy change. A large negative heat-capacity change of the binding, -156 kJ.mol(-1).K(-1), at all temperatures examined indicates that hydrophobic interaction is a major force for the binding. The binding stoichiometry shows one dimeric GAPDH intermediate per PDI monomer. The refolding of GAPDH assisted by PDI is a largely exothermic reaction at 15.0-25.0 degrees C. With increasing temperature from 15.0 to 37.0 degrees C, the PDI-assisted reactivation yield of denatured GAPDH upon dilution decreases. At 37.0 degrees C, the spontaneous reactivation, PDI-assisted reactivation and intrinsic molar enthalpy change during the PDI-assisted refolding of GAPDH are not detected.     

Thermodynamics of the binding of the C-terminal repeat domain of Streptococcus sobrinus glucosyltransferase-I to dextran

Glucosyltransferases (GTFs) secreted by mutans streptococci and some other lactic acid bacteria catalyze glucan synthesis from sucrose, and possess a C-terminal glucan-binding domain (GBD) containing homologous, directly repeating units. We prepared a series of C-terminal truncated forms of the GBD of Streptococcus sobrinus GTF-I and studied their binding to dextran by isothermal titration calorimetry. The binding of all truncates was strongly exothermic. Their titration curves were analyzed assuming that the GBD recognizes and binds to a stretch of dextran chain, not to a whole dextran molecule. Both the number of glucose units constituting the dextran stretch (n) and the accompanying enthalpy change (DeltaH degrees ) are proportional to the molecular mass of the GBD truncate, with which the Gibbs energy change calculated by the relation DeltaG degrees = -RT ln K (R, the gas constant; T, the absolute temperature; K, the binding constant of a truncate for a dextran stretch of n glucose units) also increases linearly. For the full-length GBD (508 amino acid residues), n = 33.9, K = 4.88 x 10(7) M-1, and DeltaH degrees = -289 kJ mol-1 at 25 degrees C. These results suggest that identical, independent glucose-binding subsites, each comprising 14 amino acid residues on average, are arranged consecutively from the GBD N-terminus. Thus, the GBD binds tightly to a stretch of dextran chain through the adding up of individually weak subsite/glucose interactions. Furthermore, the entropy change accompanying the GBD/dextran interaction as given by the relation DeltaS degrees = (DeltaG degrees - DeltaH degrees)/T has a very large negative value, probably because of a loss of the conformational freedom of dextran and GBD after binding.     

Thermodynamic signature of GCN4-bZIP binding to DNA indicates the role of water in discriminating between the AP-1 and ATF/CREB sites

The energetic basis of GCN4-bZIP complexes with the AP-1 and ATF/CREB sites was investigated by optical methods and scanning and isothermal titration microcalorimetry. The dissociation constant of the bZIP dimer was found to be significantly higher than that of its isolated leucine zipper domain: at 20 degrees C it is 1.45microM and increases with temperature. To avoid complications from dissociation of this dimer, DNA binding experiments were carried out using an SS crosslinked version of the bZIP. The thermodynamic characteristics of the bZIP/DNA association measured at different temperatures and salt concentrations were corrected for the contribution of refolding the basic segment upon binding, determined from the scanning calorimetric experiments. Fluorescence anisotropy titration experiments showed that the association constants of the bZIP at 20 degrees C with the AP-1 and ATF/CREB binding sites do not differ much, being 1.5nM and 6.4nM, corresponding to Gibbs energies of -49kJmol(-1) and -46kJmol(-1), respectively. Almost half of the Gibbs energy is attributable to the electrostatic component, resulting from the entropic effect of counterion release upon DNA association with the bZIP and is identical for both sites. In contrast to the Gibbs energies, the enthalpies of association of the fully folded bZIP with the AP-1 and ATF/CREB sites, and correspondingly the entropies of association, are very different. bZIP binding to the AP-1 site is characterized by a substantially larger negative enthalpy and non-electrostatic entropy than to the ATF/CREB site, implying that the AP-1 complex incorporates significantly more water molecules than the ATF/CREB complex.     

Interaction of a goose-type lysozyme with chitin oligosaccharides as determined by NMR spectroscopy

The interaction between a goose-type lysozyme from ostrich egg white (OEL) and chitin oligosaccharides [(GlcNAc)(n) (n = 2, 4 and 6)] was studied by nuclear magnetic resonance (NMR) spectroscopy. A stable isotope-labelled OEL was produced in Pichia pastoris, and backbone resonance assignments for the wild-type and an inactive mutant (E73A OEL) were achieved using modern multi-dimensional NMR techniques. NMR titration was performed with (GlcNAc)(n) for mapping the interaction sites of the individual oligosaccharides based on the shifts in the two-dimensional heteronuclear single quantum correlation (HSQC) resonances. In wild-type OEL, the interaction sites for (GlcNAc)(n) were basically similar to those determined by X-ray crystallography. In E73A OEL, however, the interaction sites were spread more widely over the substrate-binding cleft than expected, due to the multiple modes of binding. The association constant for E73A OEL and (GlcNAc)(6) calculated from the shifts in the Asp97 resonance (7.2 × 10(3) M(-1)) was comparable with that obtained by isothermal titration calorimetry (5.3 × 10(3) M(-1)). The interaction was enthalpy-driven as judged from the thermodynamic parameters (ΔH = -6.1 kcal/mol and TΔS = -1.0 kcal/mol). This study provided novel insights into the oligosaccharide binding mechanism and the catalytic residues of the enzymes belonging to family GH-23.     

Myo-inositol hexasulphate and low molecular weight heparin binding to human acidic fibroblast growth factor: a calorimetric and FTIR study

The interaction of an amino-terminal-truncated 139 amino-acids form of human acidic fibroblast growth factor with myo-inositol hexasulphate and low molecular weight (3500 g mol(-1)) heparin has been studied by isothermal titration calorimetry, differential scanning calorimetry and Fourier transform infrared spectroscopy. A slightly higher affinity for the monosaccharide has been measured. The binding of the ligands causes an increase of 13--15 degrees C in the melting temperature of the free protein (45 degrees C). From measured enthalpy and heat capacity changes, calculations of changes in accessible surface areas have been made. These calculations, together with infrared spectroscopy data, indicate that a small conformational change is induced by the binding of both ligands. This conformational change would affect the tertiary structure, not the secondary one.     

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L(-1)) than in its absence (93 micromol L(-1)). Apparent binding constant values were 2160 and 10,000 L mol(-1), respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide.     

Melittin interaction with sulfated cell surface sugars

Melittin is a 26-residue cationic peptide with cytolytic and antimicrobial properties. Studies on the action mechanism of melittin have focused almost exclusively on the membrane-perturbing properties of this peptide, investigating in detail the melittin-lipid interaction. Here, we report physical-chemical studies on an alternative mechanism by which melittin could interact with the cell membrane. As the outer surface of many cells is decorated with anionic (sulfated) glycosaminoglycans (GAGs), a strong Coulombic interaction between the two oppositely charged molecules can be envisaged. Indeed, the present study using isothermal titration calorimetry reveals a high affinity of melittin for several GAGs, that is, heparan sulfate (HS), dermatan sulfate, and heparin. The microscopic binding constant of melittin for HS is 2.4 x 10 (5) M (-1), the reaction enthalpy is Delta H melittin (0) = -1.50 kcal/mol, and the peptide-to-HS stoichiometry is approximately 11 at 10 mM Tris, 100 mM NaCl at pH 7.4 and 28 degrees C. Delta H melittin (0) is characterized by a molar heat capacity of Delta C P (0) = -227 cal mol (-1) K (-1). The large negative heat capacity change indicates that hydrophobic interactions must also be involved in the binding of melittin to HS. Circular dichroism spectroscopy demonstrates that the binding of the peptide to HS induces a conformational change to a predominantly alpha-helical structure. A model for the melittin-HS complex is presented. Melittin binding was compared with that of magainin 2 and nisin Z to HS. Magainin 2 is known for its antimicrobial properties, but it does not cause lysis of the eukaryotic cells. Nisin Z shows activity against various Gram-positive bacteria. Isothermal titration calorimetry demonstrates that magainin 2 and nisin Z do not bind to HS (5-50 degrees C, 10 mM Tris, and 100 mM NaCl at pH 7.4).     

Energetic basis of molecular recognition in a DNA aptamer

The thermal stability and ligand binding properties of the L-argininamide-binding DNA aptamer (5'-GATCGAAACGTAGCGCCTTCGATC-3') were studied by spectroscopic and calorimetric methods. Differential calorimetric studies showed that the uncomplexed aptamer melted in a two-state reaction with a melting temperature T(m)=50.2+/-0.2 degrees C and a folding enthalpy DeltaH(0)(fold)=-49.0+/-2.1 kcal mol(-1). These values agree with values of T(m)=49.6 degrees C and DeltaH(0)(fold)=-51.2 kcal mol(-1) predicted for a simple hairpin structure. Melting of the uncomplexed aptamer was dependent upon salt concentration, but independent of strand concentration. The T(m) of aptamer melting was found to increase as L-argininamide concentrations increased. Analysis of circular dichroism titration data using a single-site binding model resulted in the determination of a binding free energy DeltaG(0)(bind)=-5.1 kcal mol(-1). Isothermal titration calorimetry studies revealed an exothermic binding reaction with DeltaH(0)(bind)=-8.7 kcal mol(-1). Combination of enthalpy and free energy produce an unfavorable entropy of -TDeltaS(0)=+3.6 kcal mol(-1). A molar heat capacity change of -116 cal mol(-1) K(-1) was determined from calorimetric measurements at four temperatures over the range of 15-40 degrees C. Molecular dynamics simulations were used to explore the structures of the unligated and ligated aptamer structures. From the calculated changes in solvent accessible surface areas of these structures a molar heat capacity change of -125 cal mol(-1) K(-1) was calculated, a value in excellent agreement with the experimental value. The thermodynamic signature, along with the coupled CD spectral changes, suggest that the binding of L-argininamide to its DNA aptamer is an induced-fit process in which the binding of the ligand is thermodynamically coupled to a conformational ordering of the nucleic acid.     

Inhibition study of adenosine deaminase by caffeine using spectroscopy and isothermal titration calorimetry

Kinetic and thermodynamic studies were made on the effect of caffeine on the activity of adenosine deaminase in 50 mM sodium phosphate buffer, pH 7.5, using UV spectrophotometry and isothermal titration calorimetry (ITC). An uncompetitive inhibition was observed for caffeine. A graphical fitting method was used for determination of binding constant and enthalpy of inhibitor binding by using isothermal titration microcalorimetry data. The dissociation-binding constant is equal to 350 microM by the microcalorimetry method, which agrees well with the value of 342 microM for the inhibition constant that was obtained from the spectroscopy method. Positive dependence of caffeine binding on temperature indicates a hydrophobic interaction.