Non-polyelectrolytic Forces Research Articles

Entropically driven binding of Camptothecin in the minor groove of salmon testes DNA

The present study focuses on binding association of Camptothecin (CMT) towards natural deoxy-ribonucleic acid (salmon testes, ST) under physiological conditions of pH 7.4. Extensive spectroscopic and computational techniques have been employed to elucidate thermodynamics of the said interaction. UV and fluorescence analyses portrays significant intensity changes (hyper-chromic and hypsochromic) in the spectra, which confirms effective CMT binding to ST DNA. The McGhee-von Hipple method and Scatchard plot analyses estimated the binding affinities in 105 M−1 range. Associated thermodynamic data revealed spontaneous and exothermic nature of binding. Temperature-dependent fluorescence showed negative change in enthalpy and positive change in entropy, leading to the formation of a 1:1 adduct. Non-polyelectrolytic forces appeared to be the driving force of the ligand-DNA interaction, according to salt-dependent fluorescence. Dye displacement assay, viscosity study, DNA melting, iodide quenching, urea denaturation assay examined the minor groove nature of CMT. In silico docking study examined precise molecular representations of the minor groove binding mechanism that formed between the complex, and the study's findings were consistent with the experimental results. Simulation studies also validated the experimental analysis and docking data. These findings could expedite the process of creating new and improved CMT molecular derivatives and help in the creation of DNA-targeted medicines, which may be beneficial from a pharmaceutical point of view.

Exothermic binding and structural alteration of the carrier protein lysozyme induced by Yohimbine: A spectroscopic, calorimetric and cheminformatic insight

The crucial function of bioactive ligands in the interaction with diverse proteins has garnered significant attention in the fields of pharmacokinetics and pharmacodynamics, hence generating considerable interest in the discipline of medicinal chemistry. To better comprehend its molecular mechanism, we implored a detailed biophysical analyses (using multi-spectroscopic, calorimetric, and computational tools) of the carrier protein Lysozyme, Lyz (also known as “Muramidase”) with Yohimbine (Yoh). The binding constant (K) of the Lyz-Yoh complex was estimated to be 105 M−1, exhibiting a progressive decline as temperature increased. The thermodynamic-profiling indicated the binding to be associated with an exothermic interaction driven by entropy. The confirmation of spontaneous binding is evidenced by the observed decrease in free energy. The variation in ionic strength indicated that non-polyelectrolytic forces were involved in the association. In addition, it was investigated how different metal ions affected the Lyz-Yoh binding. Calorimetric studies revealed the binding to be exothermic in nature, which also aligns with spectroscopic data. Results from circular dichroism (CD) experiments, demonstrated the structural alterations brought about in Lyz by Yoh. The likely binding sites and protein residuals to the ligand (Yoh) were illustrated using theoretical techniques, viz., molecular docking and molecular dynamic (MD) simulation. In clinical and pharmaceutical assessments, these metrics are especially relevant for rational drug development.

Mechanistic investigation into the binding property of Yohimbe towards natural polymeric DNAs

DNA interactions with multivalent ligand(s) have increasingly become the subject of substantial research. For several small molecules with therapeutic potential, nucleic acids serve as their primary molecular target. Such interaction has been shown to affect transcription or replication, ultimately leading to apoptotic cell death. As a result, researchers are becoming increasingly interested in understanding how small molecules interact with DNA making it possible to develop new, DNA-specific drugs. The bioactive indole alkaloid, Yohimbe (Yohimbine; Yh) has been broadly studied in pharmacological properties while its binding mode to DNA has not been explicated so far. This study adopted molecular modelling and multi-spectroscopic methods to investigate the interaction between Yohimbine and herring testes (HT DNA) in physiological conditions. Minor hypochromic and bathochromic shifts of fluorescence intensity were observed, suggesting the binding of Yh to HT DNA. The Scatchard plot analyses using the McGhee-von Hipple method revealed non-cooperative binding and affinities in the range of 105 M−1. The thermodynamic parameters suggested exothermic binding, which was favoured by negative enthalpy and positive entropy changes from temperature-dependent fluorescence experiments. Salt-dependent fluorescence suggested that the interaction between the ligand and DNA was governed by non-polyelectrolytic forces. The results of iodide quenching, urea denaturation assay, dye displacement, and in silico molecular docking, suggested groove binding of Yh to HT DNA. Thus, the groove binding mechanism of interaction was validated by both biophysical and computational techniques. The structural elucidation and energetic profiling of Yh's interaction with naturally occurring polymeric DNA can be useful to the development of DNA-targeted therapeutics.

New insights into self-structure induction in poly (rA) by Quinacrine through non-classical intercalation: Spectroscopic and theoretical perspectives

Self-structure induction in a single stranded polyriboadenylic acid [poly (rA)] is an auspicious physiological phenomenon which switches off protein production in tumor cells. In the present study, the self-structure induction process in poly (rA) moiety was thoroughly investigated using various steady state and time resolved techniques. Optical melting pattern directly evidenced the formation of self-structured assembly in single stranded poly (rA) upon complexation with quinacrine. Further, UV-absorption spectroscopic studies revealed that quinacrine binds to poly (rA) in co-operative fashion and the indication of intercalative mode of binding first came out with the involvement of around two base pairs of poly (rA) in the complexation. Experimental observations established the unconventional or non-classical intercalation of quinacrine molecule inside self-structured duplex poly (rA) moiety. This complexation was accompanied with negative enthalpy change and positive entropy change; suggesting strong van der Waals and the H-bonding interactions as the major governing forces in the complexation. Moreover, ionic strength dependent binding study established that the non-polyelectrolytic forces were the dominating forces. Further, the photo physical behavior of QN was authenticated using time dependent density functional theory (TDDFT) where both the ground and excited states were exploited.

Structural Insight into Groove Binding of Yohimbine with Calf Thymus DNA: A Spectroscopic, Calorimetric, and Computational Approach.

A variety of anticancer and antibacterial drugs target DNA as one of their primary intracellular targets. Understanding ligand-DNA interactions and developing new, promising bioactive molecules for clinical use are greatly aided by elucidating the interaction between small molecules and natural polymeric DNAs. Small molecules' ability to attach to and inhibit DNA replication and transcription provides more information on how drugs impact the expression of genes. Yohimbine has been broadly studied in pharmacological properties, while its binding mode to DNA has not been explicated so far. In this study, an attempt was made to explore the interaction between Yohimbine (YH) and calf thymus (CT-DNA) by using varying thermodynamics and in silico approaches. Minor hypochromic and bathochromic shifts of fluorescence intensity were observed, suggesting the binding of YH to CT-DNA. The Scatchard plot analysis using the McGhee-von Hipple method revealed noncooperative binding and affinities in the range of 105 M-1. The binding stoichiometry value is 2:1 (2 molecules of YH were span by 1 base pair) and was determined by Job's plot. The thermodynamic parameters suggested exothermic binding, which was favored by negative enthalpy and positive entropy changes from both isothermal titration calorimetry and temperature-dependent fluorescence experiment. Salt-dependent fluorescence suggested that the interaction between the ligand and DNA was governed by nonpolyelectrolytic forces. Kinetics experiment confirmed the static type of quenching. The results of iodide quenching, urea denaturation assay, dye displacement, DNA melting, and in silico molecular docking (MD) suggested groove binding of YH to CT-DNA. Circular dichroism spectra confirmed minimal perturbation of CT-DNA with YH binding via groove region. Therefore, the groove binding mechanism of interaction was validated by biophysical techniques and in silico, MD approaches. The findings supported here may contribute to the development of new YH therapeutics possessing better efficacy and lesser side effects.

Heme Protein Binding of Sulfonamide Compounds: A Correlation Study by Spectroscopic, Calorimetric, and Computational Methods.

Protein–ligand interaction studies are useful to determine the molecular mechanism of the binding phenomenon, leading to the establishment of the structure–function relationship. Here, we report the binding of well-known antibiotic sulfonamide drugs (sulfamethazine, SMZ; and sulfadiazine, SDZ) with heme protein myoglobin (Mb) using spectroscopic, calorimetric, ζ potential, and computational methods. Formation of a 1:1 complex between the ligand and Mb through well-defined equilibrium was observed. The binding constants obtained between Mb and SMZ/SDZ drugs were on the order of 104 M–1. SMZ with two additional methyl (−CH3) substitutions has higher affinity than SDZ. Upon drug binding, a notable loss in the helicity (via circular dichroism) and perturbation of the three-dimensional (3D) protein structure (via infrared and synchronous fluorescence experiments) were observed. The binding also indicated the dominance of non-polyelectrolytic forces between the amino acid residues of the protein and the drugs. The ligand–protein binding distance signified high probability of energy transfer between them. Destabilization of the protein structure upon binding was evident from differential scanning calorimetry results and ζ potential analyses. Molecular docking presented the best probable binding sites of the drugs inside protein pockets. Thus, the present study explores the potential binding characteristics of two sulfonamide drugs (with different substitutions) with myoglobin, correlating the structural and energetic aspects.

Interaction of proflavine with the RNA polynucleotide polyriboadenylic acid–polyribouridylic acid: photophysical and calorimetric studies

The binding of proflavine, an acriflavine derivative, with the RNA polynucletodide polyadenylic acid–polyuridylic acid is investigated here to understand the structural and thermodynamic basis of the binding process. Such binding data are crucial for designing viable theraperutic agents. Spectroscopic studies clearly suggest a strong binding interaction between proflavine and polyadenylic acid–polyuridylic acid leading to efficient energy transfer between the poly AU base pairs and proflavine. The stoichiometry of proflavine polyadenylic acid–polyuridylic acid binding was independently estimated by continuous variation analysis of Job. An intercalative binding model is envisaged for the binding from hydrodynamic studies. Circular dichroism experiments revealed that the binding induced conformational changes in the RNA, and also led to induction of optical activity in the bound dye molecules. The binding affinity of the complex was deduced to be (6.57 ± 0.75) 105 M−1 at (298.15 ± 0.10) K from isothermal titration calorimetry experiment. Positive entropy and negative enthalpy changes characterized the complexation. The binding was observed to be weaker both at higher temperatures and increased [Na+]. The affinity of binding decreased with increasing [Na+]. When the Gibbs energy was parsed between polyelectrolytic and nonpolyelectropytic components, it surprisingly revealed a higher role for the non-polyelectrolytic forces. These results present new data for developing RNA targeted ligands.Communicated by Ramaswamy H. Sarma

Thermodynamic investigation of Hoechst 33258-poly(A).poly(U) binding through calorimetric studies

Abstract Interaction of the important minor groove binder Hoechst 33258 with poly(A).poly(U) was studied using isothermal titration calorimetry. The equilibrium constant of the association was calculated to be (4.00 ± 0.80) 106 M−1 at 298.15 K. The Hoechst RNA binding was driven by large negative enthalpy contributions. The equilibrium constant values for the binding reaction decreased with increasing [Na+] concentration. Parsing of the standard molar Gibbs energy change revealed that non-polyelectrolytic forces dominated the complexation process at all the salt concentrations studied. Temperature dependent calorimetric studies revealed that the equilibrium constant decreased in magnitude with increasing temperature in the range (288.15 ± 0.10)–(308.15 ± 0.10) K. Furthermore, there were significant alterations in the thermodynamic parameters with change in temperature; the binding became increasingly enthalpy driven and the standard molar entropic contribution decreased in magnitude and became unfavourable as the temperature was increased. Negative heat capacity change and an enthalpy-entropy compensation phenomenon were also observed. The data observed may be useful for developing structure selective RNA-based therapeutics.

Thermodynamic analysis of the complexation of quinacrine with tRNAPhe

Abstract The thermodynamics of the interaction of the acridine derivative quinacrine with tRNA Phe was studied using isothermal titration calorimetry. The binding was monophasic and exothermic. At (298.15 ± 0.01) K the equilibrium constant was calculated to be (1.76 ± 0.70)·10 5 M −1 . The binding stoichiometry was (0.15 ± 0.04). The binding was predominantly enthalpy driven (Δ H o = −28.39 ± 0.60 kJ·mol −1 ). Salt dependent ITC studies followed by dissection of the standard molar Gibbs energy change showed that the binding reaction was driven by both polyelectrolytic and non-polyelectrolytic forces with the non-polyelectrolytic forces being dominant. Temperature dependent ITC studies showed that the binding became increasingly enthalpy driven with rise in temperature. The binding was characterized by negative standard molar heat capacity change and enthalpy-entropy compensation behaviour.

Exploring the binding interaction of potent anticancer drug topotecan with human serum albumin: spectroscopic, calorimetric and fibrillation study

This manuscript describes the interaction of the topoisomerase I inhibitor anticancer drug topotecan with human serum albumin using microcalorimetry, circular dichroism, and atomic force microscopy imaging techniques. Conformational change in albumin was ascertained from circular dichroism and synchronous fluorescence study that revealed a small but definitive partial unfolding of the protein structure upon drug binding. Isothermal titration calorimetry study indicated a favorable exothermic interaction with a binding affinity of the order of ~105 M−1 at 293.15 K. A 1:1 binding stoichiometry was established. The binding reaction was largely enthalpy dominated with negative standard molar Gibbs energy change. Ionic strength variation study revealed that non-polyelectrolytic forces played dominant role in the interaction and remained almost invariant at all salt concentrations. Upon complex formation, the stabilization of the protein structure against thermal denaturation occurred. Atomic force microscopy study enabled imaging of fibrils of the protein and its complex with topotecan.

Binding and Inhibitory Effect of the Dyes Amaranth and Tartrazine on Amyloid Fibrillation in Lysozyme.

Interaction of two food colorant dyes, amaranth and tartrazine, with lysozyme was studied employing multiple biophysical techniques. The dyes exhibited hypochromic changes in the presence of lysozyme. The intrinsic fluorescence of lysozyme was quenched by both dyes; amaranth was a more efficient quencher than tartrazine. The equilibrium constant of amaranth was higher than that of tartarzine. From FRET analysis, the binding distances for amaranth and tartrazine were calculated to be 4.51 and 3.93 nm, respectively. The binding was found to be dominated by non-polyelectrolytic forces. Both dyes induced alterations in the microenvironment surrounding the tryptophan and tyrosine residues of the protein, with the alterations being comparatively higher for the tryptophans than the tyrosines. The interaction caused significant loss in the helicity of lysozyme, the change being higher with amaranth. The binding of both dyes was exothermic. The binding of amaranth was enthalpy driven, while that of tartrazine was predominantly entropy driven. Amaranth delayed lysozyme fibrillation at 25 μM, while tartrazine had no effect even at 100 μM. Nevertheless, both dyes had a significant inhibitory effect on fibrillogenesis. The present study explores the potential antiamyloidogenic property of these azo dyes used as food colorants.

Probing the binding of anticancer drug topotecan with human hemoglobin: Structural and thermodynamic studies

Protein - ligand interactions play pivotal role in almost all the biological processes occurring in living organisms, and therefore such studies hold immense importance from the standpoint of rational drug design and development. In this study the binding of the topoisomerase I inhibitor drug, topotecan to hemoglobin was probed using various biophysical and microcalorimetry techniques. Spectrofluorimetric data confirmed the static nature of the quenching mechanism of the protein induced by the drug. Significant conformational changes in the protein were ascertained from circular dichroism and three dimensional fluorescence results. Synchronous fluorescence study revealed an increase in the polarity around the Trp residues of the protein while atomic force microscopy study enabled to obtain images of the bound molecules. Isothermal titration calorimetry studies indicated an exothermic binding with a negative Gibbs energy change; ionic strength variation suggested a greater contribution from non-polyelectrolytic forces in the binding process. Differential scanning calorimetry studies indicated an increased thermal stabilization of the protein upon topotecan binding which is also in close agreement with the results obtained from absorbance and circular dichroism melting studies. Overall this manuscript presents results on the molecular interaction from structural and energetic perspectives providing an in depth insight into drug-protein interaction.

Multispectroscopic and calorimetric studies on the binding of the food colorant tartrazine with human hemoglobin

Interaction of the food colorant tartrazine with human hemoglobin was studied using multispectroscopic and microcalorimetric techniques to gain insights into the binding mechanism and thereby the toxicity aspects. Hemoglobin spectrum showed hypochromic changes in the presence of tartrazine. Quenching of the fluorescence of hemoglobin occurred and the quenching mechanism was through a static mode as revealed from temperature dependent and time-resolved fluorescence studies. According to the FRET theory the distance between β-Trp37 of hemoglobin and bound tartrazine was evaluated to be 3.44nm. Synchronous fluorescence studies showed that tartrazine binding led to alteration of the microenvironment around the tryptophans more in comparison to tyrosines. 3D fluorescence and FTIR data provided evidence for conformational changes in the protein on binding. Circular dichroism studies revealed that the binding led to significant loss in the helicity of hemoglobin. The esterase activity assay further complemented the circular dichroism data. Microcalorimetric study using isothermal titration calorimetry revealed the binding to be exothermic and driven largely by positive entropic contribution. Dissection of the Gibbs energy change proposed the protein-dye complexation to be dominated by non-polyelectrolytic forces. Negative heat capacity change also corroborated the involvement of hydrophobic forces in the binding process.

Coralyne induced self-structure in polyadenylic acid: Thermodynamics of the structural reorganization

Thermodynamics of the induction of self-structure in polyadenylic acid by the cytotoxic isoquinoline alkaloid coralyne was investigated using calorimetry tools. The binding was an exothermic process driven by large negative enthalpy change. The equilibrium constant at [K+]=(130±0.01)mM and T=(298.15±0.01)K was calculated to be (1.34±0.07)·106M−1. Temperature dependent calorimetric studies suggested weakening of the self-structure at higher temperatures. The self-structure induction process was characterized by complete enthalpy-entropy compensation. The temperature dependence of the enthalpy change yielded negative heat capacity value. The equilibrium constant decreased with increasing [K+] apparently due to the weakening of the self-structure at higher salt concentrations. Differential scanning calorimetry studies testified for the formation of self-structure in (50±0.01)–(130±0.01)mM [K+]. Parsing of the standard molar Gibbs energy change revealed that the non-polyelectrolytic forces dominated the self-structure induction process.

Thermodynamics of the induction of self-structure in polyadenylic acid by proflavine

The energetics of self-structure induction in polyadenylic acid in the presence of proflavine has been studied using isothermal titration calorimetry. The self-structure induction process was exothermic and driven by large positive entropy change. The equilibrium constant at T=(298.15±0.01)K and [Na+]=(130±0.01)mM was calculated to be (1.01±0.08)·106M−1. Salt dependent calorimetric studies revealed that the equilibrium constant increased with increasing [Na+] in the 50–130mM range suggesting enhanced binding preference at higher salt concentrations, apparently due to easier self-structure formation at higher [Na+]. Dissection of the standard molar Gibbs energy change clearly established that the self-structure induction was driven by non-polyelectrolytic forces and the polyelectrolytic contribution was relatively small. The equilibrium constant decreased with increasing temperature indicating destabilization of the self-structure at higher temperatures. Negative standard molar heat capacity value obtained from the temperature dependence of the enthalpy change suggested that hydrophobic forces are important for the self-structure induction. Furthermore, a complete enthalpy–entropy compensation phenomenon was also observed.

Calorimetric investigation on the interaction of proflavine with human telomeric G-quadruplex DNA

The interaction of an acridine dye, proflavine, with human telomeric G-quadruplex DNA was characterized by isothermal titration calorimetry and differential scanning calorimetry. The equilibrium constant of binding was deduced to be (1.36±0.09)·106M−1 at T=298.15K. The binding reaction was driven by both negative enthalpy and positive entropy contributions. The equilibrium constant decreased and the reaction became increasingly enthalpy driven with rise in temperature. However, the standard molar Gibbs energy change exhibited only marginal alterations suggesting the occurrence of enthalpy–entropy compensation. Negative heat capacity values were also obtained from the temperature dependence of enthalpy change. Parsing of the standard molar Gibbs energy using the salt dependent calorimetric data revealed that the binding was dominated by non-polyelectrolytic forces which remained virtually unaltered with changing salt concentration. Proflavine binding also significantly enhanced the thermal stability of G-quadruplex DNA against thermal unfolding.

A microcalorimetric study on the binding of proflavine with tRNAphe

The interaction of proflavine with tRNAphe was studied using isothermal titration calorimetry to gain insights into thermodynamics of the binding reaction. Isothermal titration calorimetric studies revealed the association to be exothermic and favored by both negative enthalpy and positive entropy changes. The equilibrium constant for the complexation process was calculated to be (2.85±0.09) 105M−1 at T=293.15K. The equilibrium constant decreased with increasing temperature suggesting that the complexation is less favored at higher temperatures. The standard molar enthalpic contribution enhanced in magnitude while the standard molar entropic contribution decreased with increasing temperature. The complexation was further characterized by negative standard molar heat capacity and (enthalpy+entropy) compensation. Salt dependent ITC studies and parsing of the standard molar Gibbs energy revealed that the complexation process was largely dominated by non-polyelectrolytic forces which remained almost unaltered at all the salt concentrations studied.

A thermodynamic investigation on the binding of phenothiazinium dyes azure A and azure B to double stranded RNA polynucleotides

The thermodynamics of the reactions of the two phenothiazinium dyes azure A and azure B with the three double stranded ribonucleic acids, poly(A).poly(U), poly(C).poly(G), poly(I).poly(C) were investigated using DSC and ITC. The bound dyes stabilized the RNAs against thermal strand separation. The binding of azure A to the RNAs was predominantly enthalpy dominated while the binding of azure B was favoured by both negative enthalpy and favourable entropy changes. Although electrostatic interaction had a significant role in the binding, non-polyelectrolytic forces dominated the binding process. The negative values of heat capacity changes for the binding suggested a substantial hydrophobic contribution to the binding process. The overall binding affinity of both the dyes to the RNAs varied in the order, poly(A).poly(U)>poly(C).poly(G)>poly(I).poly(C).

Thermodynamic characterization of proflavine–DNA binding through microcalorimetric studies

The interaction of an important acridine dye, proflavine hydrochloride, with double stranded DNA was investigated using isothermal titration calorimetry and differential scanning calorimetry. The equilibrium constant for the binding reaction was calculated to be (1.60±0.04)·105·M−1 at T=298.15K. The binding of proflavine hydrochloride to DNA was favored by both negative enthalpy and positive entropy contributions to the Gibbs energy. The equilibrium constant for the binding reaction decreased with increasing temperature. The standard molar enthalpy change became increasingly negative while the standard molar entropy change became less positive with rise in temperature. However, the standard molar Gibbs free energy change varied marginally suggesting the occurrence of enthalpy–entropy compensation phenomenon. The binding reaction was dominated by non-polyelectrolytic forces which remained virtually unchanged at all the salt concentrations studied. The binding also significantly increased the thermal stability of DNA against thermal denaturation.

Thermodynamics of the interaction of the food additive tartrazine with serum albumins: A microcalorimetric investigation

The thermodynamics of the interaction of the food colourant tartrazine with two homologous serum proteins, HSA and BSA, were investigated, employing microcalorimetric techniques. At T=298.15K the equilibrium constants for the tartrazine-BSA and HSA complexation process were evaluated to be (1.92±0.05)×105M−1 and (1.04±0.05)×105M−1, respectively. The binding was driven by a large negative standard molar enthalpic contribution. The binding was dominated essentially by non-polyelectrolytic forces which remained largely invariant at all salt concentrations. The polyelectrolytic contribution was weak at all salt concentrations and accounted for only 6–18% of the total standard molar Gibbs energy change in the salt concentration range 10–50mM. The negative standard molar heat capacity values, in conjunction with the enthalpy–entropy compensation phenomenon observed, established the involvement of dominant hydrophobic forces in the complexation process. Tartrazine enhanced the stability of both serum albumins against thermal denaturation.