Protoberberine Alkaloids Berberine,Palmatine, and Coralyne Binding to Poly(dT)⋅(Poly(dA)⋅Poly(dT)) Triplex: Comparative Structural Aspects and Energetics Profiles of the Interaction

The interaction of bioactive protoberberine alkaloids berberine, palmatine, and coralyne with the DNA triplex poly(dT)⋅(poly(dA)⋅poly(dT)) was studied using biophysical and calorimetric techniques. All three alkaloids bound the triplex cooperatively. Berberine and palmatine predominantly stabilized the triplex structure, while coralyne stabilized both triplex and duplex structures as inferred from optical thermal melting profiles. Fluorescence quenching, polarization, and viscometric studies hinted at an intercalative mode of binding for the alkaloids to the triplex, coralyne being more strongly intercalated compared to partial intercalation of berberine and palmatine. The overall affinity of coralyne was two order higher (2.29×10⁷ M ⁻¹) than that of berberine (3.43×10⁵ M ⁻¹) and palmatine (2.34×10⁵ M ⁻¹). Isothermal titration calorimetric studies revealed that the binding to the triplex was favored by negative enthalpy change (ΔH=−3.34 kcal/mol) with favorable entropy contribution (TΔS = 4.07 kcal/mol) for berberine, favored by almost equal negative enthalpy (ΔH =−3.88 kcal/mol) and entropy changes (TΔS = 3.37 kcal/mol) for palmatine, but driven by large enthalpy contributions (ΔH =−25.62 kcal/mol and TΔS =−15.21 kcal/mol) for coralyne. These results provide new insights on the binding of isoquinoline alkaloids to the DNA triplex structure.     

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.     

Thermodynamics of the binding of cytotoxic protoberberine molecule coralyne to deoxyribonucleic acids

The binding thermodynamics of the interaction of protoberberine molecule coralyne to various DNAs have been investigated. Thermodynamic data revealed that the binding was enthalpy driven in GC rich DNA and GC polynucleotides while the same was favored by both negative enthalpy and positive entropy changes in the AT rich DNA and AT polymers. Parsing the free energy change of the binding in terms of polyelectrolytic and nonpolyelectrolytic contribution showed the involvement of major contributions from the later. The heat capacity change (DeltaC(p) degrees ) for the binding of coralyne to calf thymus DNA and Micrococcus lysodeikticus DNA was - 147 and - 190cal/(mol K) respectively. The binding data in these systems also showed significant enthalpy-entropy compensation confirming the involvement of multiplicity of weak non-covalent interactions in agreement with the negative heat capacity data. Circular dichroic studies revealed that the binding was accompanied by moderate conformational change of B-form structure and more importantly the achiral alkaloid molecules acquired strong induced optical activity. These results contribute to the understanding of energetics of coralyne-DNA complexation that will guide synthetic efforts of medicinal chemists for developing better therapeutic agents.     

RNA targeting by DNA binding drugs: Structural,conformational and energetic aspects of the binding of quinacrine and DAPI to A-form and H-form of poly(rC)·poly(rG)

A key step in the rational design of new RNA binding small molecules necessitates a complete elucidation of the molecular aspects of the binding of existing molecules to RNA structures. This work focuses towards the understanding of the interaction of a DNA intercalator, quinacrine and a minor groove binder 4′,6-diamidino-2-phenylindole (DAPI) with the right handed Watson–Crick base paired A-form and the left-handed Hoogsteen base paired H^L-form of poly(rC)·poly(rG) evaluated by multifaceted spectroscopic and viscometric techniques. The energetics of their interaction has also been elucidated by isothermal titration calorimetry. Results of this study converge to suggest that (i) quinacrine intercalates to both A-form and H^L-form of poly(rC)·poly(rG); (ii) DAPI shows both intercalative and groove-binding modes to the A-form of the RNA but binds by intercalative mode to the H^L-form. Isothermal calorimetric patterns of quinacrine binding to both the forms of RNA and of DAPI binding to the H^L-form are indicative of single binding while the binding of DAPI to the A-form reveals two kinds of binding. The binding of both the drugs to both conformations of RNA is exothermic; while the binding of quinacrine to both conformations and DAPI to the A-form (first site) is entropy driven, the binding of DAPI to the second site of A-form and H^L-conformation is enthalpy driven. Temperature dependence of the binding enthalpy revealed that the RNA–ligand interaction reactions are accompanied by small heat capacity changes that are nonetheless significant. We conclude that the binding affinity characteristics and energetics of interaction of these DNA binding molecules to the RNA conformations are significantly different and may serve as data for the development of effective structure selective RNA-based antiviral drugs.     

Interaction of isoquinoline alkaloid palmatine with deoxyribonucleic acids: binding heterogeneity, and conformational and thermodynamic aspects

The binding heterogeneity, conformational aspects, and energetics of the interaction of the cytotoxic plant alkaloid palmatine have been studied with various natural and synthetic DNAs. The alkaloid binds to calf thymus and Escherichia coli DNA that have mixed AT and GC sequences in almost equal proportions with positive cooperativity, while, with Clostridium perfringens and Micrococcus lysodeikticus DNA with predominantly high AT and GC sequences, respectively, noncooperative binding was observed. On further investigation with synthetic DNAs, the binding was observed to be cooperative with polymers like poly(dA).poly(dT) and poly(dG).poly(dC) having poly(purine)poly(pyrimidine) sequences, while with polymers poly(dA-dT).poly(dA-dT), poly(dA-dC).poly(dG-dT) and poly(dG-dC).poly(dG-dC), which have alternating purine-pyrimidine sequences, a non-cooperative binding phenomenon was observed. This suggests the binding heterogeneity of palmatine to the two types of sequences of base pairs. Circular dichroism (CD) studies revealed that the binding induced conformational changes in all the DNAs, but more importantly, the bound alkaloid molecules acquired induced optical activity, and the extent was dependent on the AT content and showed AT base-pair specificity. Energetics of the interaction of the alkaloid studied by highly sensitive isothermal titration calorimetry revealed that the binding was in most cases exothermic and favored by both enthalpy and entropy changes, while, in the case of the homo and hetero AT polymers, the same was predominantly entropy-driven. This study defines base-pair-dependent heterogeneity, conformational aspects, and energetics of palmatine binding to DNA.     

Binding of protoberberine alkaloid coralyne with double stranded poly(A): a biophysical study

Recognition of double stranded ribonucleic acid is a critical event in many biological pathways such as trafficking, editing and maturation of mRNA, interferon antiviral response and RNA interference. In the context of probing double stranded RNA binding small molecules, the interaction of the antitumor protoberberine alkaloid coralyne with double stranded poly(A) has been studied by various biophysical techniques. Typical hypochromic and bathochromic shifts in the absorption spectrum and appreciable quenching of the intrinsic fluorescence of coralyne indicated the strong affinity of coralyne to poly(A). The corresponding intrinsic binding constant evaluated from Scatchard analysis was in the order of 10(5) M(-1). The strong binding was further characterized by significant polarization of the alkaloid fluorescence and stabilization of poly(A) helix against thermal strand separation. The binding process was manifested by remarkable perturbation of the intrinsic circular dichroic spectrum of poly(A) with concomitant generation of optical activity in the bound alkaloid molecules that are otherwise achiral. Job plot analysis showed the binding stoichiometry of the interaction process to be two base pairs per alkaloid molecule. The energetics of the strong interaction was studied by isothermal titration and differential scanning calorimetric techniques that suggested the binding to be exothermic and favoured by both negative enthalpy and positive entropy changes. All these results, together with the Stern-Volmer quenching experiment in fluorescence, revealed the molecular details of the intercalation of coralyne into poly(A) duplex leading to its potential use as an agent in gene regulation in eukaryotic cells.     

RNA specific molecules: cytotoxic plant alkaloid palmatine binds strongly to poly(A)

The cytotoxic plant alkaloid palmatine was found to bind strongly by partial intercalation to single stranded poly(A) structure with binding affinity (Ka) of (8.36+/-0.26) x 10(5) M(-1). The binding of palmatine was characterized to be exothermic and enthalpy driven with one palmatine for every two adenine residues. On the other hand, the binding to the double stranded poly(A) has been found to be significantly weak. This study identifies poly(A) as a potential bio-target for the alkaloid palmatine and its use as a lead compound in antitumor drug screening.     

Recognition of human telomeric G‐quadruplex DNA by berberine analogs: effect of substitution at the 9 and 13 positions of the isoquinoline moiety

G‐quadruplex forming sequences are widely distributed in human genome and serve as novel targets for regulating gene expression and chromosomal maintenance. They offer unique targets for anticancer drug development. Here, the interaction of berberine (BC) and two of its analogs bearing substitution at 9 and 13‐position with human telomeric G‐quadruplex DNA sequence has been investigated by biophysical techniques. Both the analogs exhibited several‐fold higher binding affinity than berberine. The Scatchard binding isotherms revealed non‐cooperative binding. 9‐ω‐amino hexyl ether analog (BC1) showed highest affinity (1.8 × 10⁶ M^?1) while the affinity of the 13‐phenylpropyl analog (BC2) was 1.09 × 10⁶ M^?1. Comparative fluorescence quenching and polarization anisotropy of the emission spectra gave evidence for a stronger stacking interaction of the analogs compared to berberine. The thiazole orange displacement assay has clearly established that the analogs were more effective in displacing the end stacked dye in comparison to berberine. However, the binding of the analogs did not induce any major structural perturbation in the G‐quadruplex structure, but led to higher thermal stability. Energetics of the binding indicated that the association of the analogs was exothermic and predominantly entropy driven phenomenon. Increasing the temperature resulted in weaker binding; the enthalpic contribution increased and the entropic contribution decreased. A small negative heat capacity change with significant enthalpy–entropy compensation established the involvement of multiple weak noncovalent interactions in the binding process. The 9‐ω‐amino hexyl ether analog stabilized the G‐quadruplex structure better than the 13‐phenyl alkyl analog. Copyright © 2015 John Wiley & Sons, Ltd.     

Salt bridge exchange binding mechanism between streptavidin and its DNA aptamer – thermodynamics and spectroscopic evidences

Protein‐nucleic acids binding driven by electrostatic interactions typically are characterized by the release of counter ions, and the salt‐inhibited binding association constant (K_a) and the magnitude of exothermic binding enthalpy (ΔH). Here, we report a non‐classical thermodynamics of streptavidin (SA)–aptamer binding in NaCl (140–350 mM) solutions near room temperatures (23–27 °C). By using isothermal titration calorimetry (ITC) and circular dichroism (CD)/fluorescence spectroscopy, we found that the binding was enthalpy driven with a large entropy cost (ΔH ?20.58 kcal mol^?1, TΔS ?10.99 kcal mol^?1, and K_a 1.08 × 10⁷ M^?1 at 140 mM NaCl 25 °C). With the raise of salt concentrations, the ΔH became more exothermic, yet the K_a was almost unchanged (ΔH ?26.29 kcal mol^?1 and K_a 1.50 × 10⁷ M^?1 at 350 mM NaCl 25 °C). The data suggest that no counter Na⁺ was released in the binding. Spectroscopy data suggest that the binding, with a stoichiometry of 2, was accompanied with substantial conformational changes on SA, and the changes were insensitive to the variation of salt concentrations. To account for the non‐classical results, we propose a salt bridge exchange model. The intramolecular binding‐site salt bridge(s) of the free SA and the charged phosphate group of aptamers re‐organize to form the binding complex by forming a new intermolecular salt bridge(s). The salt bridge exchange binding process requires minimum amount of counter ions releasing but dehydration of the contacting surface of SA and the aptamer. The energy required for dehydration is reduced in the case of binding solution with higher salt concentration and account for the higher binding exothermic mainly. Copyright © 2013 John Wiley & Sons, Ltd.     

Drug‐binding energetics of human α‐1‐acid glycoprotein assessed by isothermal titration calorimetry and molecular docking simulations

This study utilizes sensitive, modern isothermal titration calorimetric methods to characterize the microscopic thermodynamic parameters that drive the binding of basic drugs to α‐1‐acid glycoprotein (AGP) and thereby rationalize the thermodynamic data in relation to docking models and crystallographic structures of the drug–AGP complexes. The binding of basic compounds from the tricyclic antidepressant series, together with miaserine, chlorpromazine, disopyramide and cimetidine, all displayed an exothermically driven binding interaction with AGP. The impact of protonation/deprotonation events, ionic strength, temperature and the individual selectivity of the A and F1*S AGP variants on drug‐binding thermodynamics was characterized. A correlation plot of the thermodynamic parameters for all of the test compounds revealed that an enthalpy–entropy compensation is in effect. The exothermic binding energetics of the test compounds were driven by a combination of favorable (negative) enthalpic (?Hº) and favorable (positive) entropic (?Sº) contributions to the Gibbs free energy (?Gº). Collectively, the data imply that the free energies that drive drug binding to AGP and its relationship to drug serum residency evolve from the complex interplay of enthalpic and entropic forces from interactions with explicit combinations of hydrophobic and polar side‐chain sub‐domains within the multi‐lobed AGP ligand binding cavity.Copyright © 2012 John Wiley & Sons, Ltd.     

Molecular recognition of DNA by small molecules: AT base pair specific intercalative binding of cytotoxic plant alkaloid palmatine

The base dependent binding of the cytotoxic alkaloid palmatine to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by competition dialysis, spectrophotometric, spectrofluorimetric, thermal melting, circular dichroic, viscometric and isothermal titration calorimetric (ITC) studies. Binding of the alkaloid to various polynucleotides was dependent upon sequences of base pairs. Binding data obtained from absorbance measurements according to neighbour exclusion model indicated that the intrinsic binding constants decreased in the order poly(dA).poly(dT)>poly(dA-dT).poly(dA-dT)>poly(dG-dC).poly(dG-dC)>poly(dG).poly(dC). This affinity was also revealed by the competition dialysis, increase of steady state fluorescence intensity, increase in fluorescence quantum yield, stabilization against thermal denaturation and perturbations in circular dichroic spectrum. Among the polynucleotides, poly(dA).poly(dT) showed positive cooperativity at binding values lower than r=0.05. Viscosity studies revealed that in the strong binding region, the increase of contour length of DNA depended strongly on the sequence of base pairs being higher for AT polymers and induction of unwinding-rewinding process of covalently closed superhelical DNA. Isothermal titration calorimetric data showed a single entropy driven binding event in the AT homo polymer while that with the hetero polymer involved two binding modes, an entropy driven strong binding followed by an enthalpy driven weak binding. These results unequivocally established that the alkaloid palmatine binds strongly to AT homo and hetero polymers by mechanism of intercalation.     

Binding affinity and in vitro cytotoxicity of harmaline targeting different motifs of nucleic acids: An ultimate drug designing approach

The work focuses towards interaction of harmaline, with nucleic acids of different motifs by multispectroscopic and calorimetric techniques. Findings of this study suggest that binding constant varied in the order single‐stranded (ss) poly(A) > double‐stranded calf thymus (CT) DNA > double‐stranded poly(G)·poly(C) > clover leaf tRNA^Phe. Prominent structural changes of ss poly(A), CT DNA, and poly(G)· poly(C) with concomitant induction of optical activity in the bound achiral alkaloid molecule was observed, while with tRNA^Phe, very weak induced circular dichroism perturbation was seen. The interaction was predominantly exothermic, enthalpy driven, and entropy favored with CT DNA and poly(G)·poly(C), while it was entropy driven with poly(A) and tRNA^Phe. Intercalated state of harmaline inside poly(A), CT DNA, and poly(G)·poly(C) was shown by viscometry, ferrocyanide quenching, and molecular docking. All these findings unequivocally pointed out preference of harmaline towards ss poly(A) inducing self‐structure formation. Furthermore, harmaline administration caused a significant decrease in proliferation of HeLa and HepG₂ cells with GI₅₀ of 28μM and 11.2μM, respectively. Nucleic acid fragmentation, cellular ultramorphological changes, decreased mitochondrial membrane potential, upregulation of p53 and caspase 3, generation of reactive oxygen species, and a significant increase in the G₂/M population made HepG₂ more prone to apoptosis than are HeLa cells.     

Interaction of aloe active compounds with calf thymus DNA

Natural anthraquinone compounds have emerged as potent anticancer chemotherapeutic agents because of their promising DNA‐binding properties. Aloe vera is among one of the very well‐known medicinal plants, and the anthraquinone derivatives like aloe emodin (ALM), aloins (ALN), and aloe emodin‐8‐glucoside (ALMG) are known to have immense biological activities. Here, we have used biophysical methods to elucidate the comparative DNA‐binding abilities of these three molecules. Steady‐state fluorescence study indicated complexation between calf thymus DNA (ctDNA) and both the molecules ALM and ALMG whereas ALN showed very weak interaction with DNA. Displacement assays with ctDNA‐bound intercalator (ethidium bromide) and a groove binder (Hoechst 33258) indicated preferential binding of both ALM and ALMG to minor groove of DNA. Isothermal titration calorimetric (ITC) data suggested spontaneous exothermic single binding mode of both the molecules: ALM and ALMG. Entropy is the most important factor which contributed to the standard molar Gibbs energy associated with relatively small favorable enthalpic contribution. The equilibrium constants of binding to ctDNA were (6.02 ± 0.10) × 10⁴ M^?1 and (4.90 ± 0.11) × 10⁴ M^?1 at 298.15 K, for ALM and ALMG, respectively. The enthalpy vs temperature plot yielded negative standard molar heat capacity value, and a strong negative correlation between enthalpy and entropy terms was observed which indicates the enthalpy entropy compensation behavior in both systems. All these thermodynamic phenomena indicate that hydrophobic force is the key factor which is involved in the binding process. Moreover, the enhancement of thermal stability of DNA helix by ALM and ALMG fully agreed to the complexation of these molecules with DNA.     

Lysozyme binding with sulfa group of antibiotics: Comparative binding thermodynamics and computational study

Protein–drug binding study addresses a broad domain of biological problems associating molecular functions to physiological processes composing and modifying safe and coherent drug therapeutics. Comparison of the binding and thermodynamic aspect of sulfa drugs, sulfamethazine (SMZ) and sulfadiazine (SDZ) with the protein, lysozyme (Lyz) was carried out using spectroscopic, molecular docking, and dynamic simulation studies. The fluorescence quenching and apparent binding constant for the binding reaction were calculated to be in the order of 10⁴ M⁻¹, slightly higher for SMZ as compared to that of SDZ and the binding stoichiometry values show 1:1 drug binding with each protein molecule. The binding was an enthalpy-driven spontaneous exothermic reaction favored by a negative enthalpy and a positive entropy contribution for both the complexes. The binding from the fluorescence quenching data suggests a static quenching mechanism dominated by non-polyelectrolytic components. Synchronous fluorescence denoted a conformational change in the tryptophan moiety of the protein. Molecular docking and dynamic simulation study provided a clearer view of the interaction pattern, where the drug resides on the binding pocket of the protein structure. Overall the protein, Lyz binding of SMZ was slightly more favored over SDZ.     

Thermodynamics of partitioning of substance P in isotropic bicelles

The temperature dependence of the partition of a neuropeptide, substance P (SP), in isotropic (q = 0.5) bicelles was investigated by using pulsed field gradient NMR diffusion technique. The partition coefficient decreases as the temperature is increased from 295 to 325 K, indicating a favorable (negative) enthalpy change upon partitioning of the peptide. Thermodynamic analysis of the data shows that the partitioning of SP at 300 K is driven by the enthalpic term (ΔH) with the value of ? 4.03 kcal mol^?1, while it is opposed by the entropic term (?TΔS) by approximately 1.28 kcal mol^?1 with a small negative change in heat capacity (ΔC_p). The enthalpy‐driven process for the partition of SP in bicelles is the same as in dodecylphosphocholine (DPC) micelles, however, the negative entropy change in bicelles of flat bilayer surface is in sharp contrast with the positive entropy change in DPC micelles of highly curved surface, indicating that the curvature of the membrane surface might play a significant role in the partitioning of peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

A comparative spectroscopic and calorimetric investigation of the interaction of amsacrine with heme proteins,hemoglobin and myoglobin

The binding of the anilido aminoacridine derivative amsacrine with the heme proteins, hemoglobin, and myoglobin, was characterized by various spectroscopic and calorimetric methods. The binding affinity to hemoglobin was (1.21?±?.05) × 10⁵ M^?1, while that to myoglobin was three times higher (3.59?±?.15) × 10⁵ M^?1. The temperature-dependent fluorescence study confirmed the formation of ground-state complexes with both the proteins. The stronger binding to myoglobin was confirmed from both spectroscopic and calorimetric studies. The binding was exothermic in both cases at the three temperatures studied, and was favored by both enthalpy and entropy changes. Circular dichroism results, three-dimensional (3D) and synchronous fluorescence studies confirmed that the binding of amsacrine significantly changed the secondary structure of hemoglobin, while the change in the secondary structure of myoglobin was much less. New insights, in terms of structural and energetic aspects of the interaction of amsacrine with the heme proteins, presented here may help in understanding the structure-activity relationship, therapeutic efficacy, and drug design aspects of acridines.     

Molecular aspects on the specific interaction of cytotoxic plant alkaloid palmatine to poly(A)

The interaction of the protoberberine alkaloid palmatine with single and double stranded structures of poly(A) was studied by various biophysical techniques. Comparative binding studies were also performed with double stranded DNA, t-RNA, poly(C)·poly(G), poly(U) and poly(C). The results of competition dialysis, fluorescence, and absorption spectral studies converge to reveal the molecular aspects of the strong and specific binding of palmatine to single stranded poly(A). The binding affinity of palmatine to natural DNA, t-RNA and double stranded poly(A) was weaker while no binding was apparent with single stranded poly(U), poly(C) and double stranded poly(C)·poly(G). The strong affinity of the alkaloid to single stranded poly(A) in comparison to the double stranded structure was also revealed from circular dichroic and viscometric studies. The effect of [Na⁺] ion concentration on the binding process revealed the significant role of electrostatic forces in the complexation. The presence of bound alkaloid also remarkably affected denaturation–renaturation of stacked helical poly(A). The energetics of the strong binding to poly(A) was studied from thermodynamic estimation from van Hoff’ analysis of the temperature dependent binding constants and ultra sensitive isothermal titration calorimertry, both suggesting the binding to be exothermic and enthalpy driven. This study provides detailed insight into the binding specificity of the natural alkaloid to single stranded poly(A) over several other single and double stranded nucleic acid structures suggesting its potential as a lead compound for RNA based drug targeting.     

Thermodynamic studies on the interaction between sodium n-dodecyl sulphate and histone H2B

The thermodynamic parameters for the interaction of the anionic detergent sodium n-dodecyl sulphate (SDS) with H2B at pH 3.2, 6.4 and 10 have been measured at 27 degrees C and 37 degrees C by equilibrium dialysis to determine the Gibbs energies of detergent binding. The data have been used to obtain the enthalpy of interaction from the temperature dependence of the equilibrium constants from the Van't Hoff relation. The enthalpy of interaction between H2B and SDS is endothermic at pH 3.2, 6.4 and 10. The shapes of the enthalpy curves at pH 3.2 and 10 show some small exothermic contribution which probably indicates folding of H2B. The interactions of H2B-SDS are dominated by the increase in entropy on detergent binding. The larger negative free energy, enthalpy and entropy changes at pH 6.4 are consistent with greater denaturation relative to pH 3.2 and 10.     

RNA binding small molecules: studies on t-RNA binding by cytotoxic plant alkaloids berberine, palmatine and the comparison to ethidium

The interaction of two natural protoberberine plant alkaloids berberine and palmatine with t-RNA(phe) was studied using various biophysical techniques and the data was compared with the binding of the classical DNA intercalator, ethidium. The results of optical thermal melting, differential scanning calorimetry and circular dichroism characterized the native cloverleaf structure of t-RNA under the conditions of the study. The strong binding of the alkaloids and ethidium to t-RNA was revealed from the absorption and fluorescence studies. The salt dependence of the binding constants enabled the dissection of the binding free energy to electrostatic and non-electrostatic contributions. This analysis revealed a surprisingly large favourable component of the non-electrostatic contribution to the binding of these charged alkaloids and ethidium to t-RNA. Isothermal titration calorimetric studies revealed that the binding of both the alkaloids is driven by a moderately favourable enthalpy decrease and a moderately favourable entropy increase while that of ethidium is driven by a large favourable enthalpy decrease. Taken together, the results suggest that the binding of these alkaloid molecules on the t-RNA structure appears to be mostly by partial intercalation while ethidium intercalates to the t-RNA. These results reveal the molecular aspects on the interaction of these alkaloids to t-RNA.