Triblock-Copolymer-Assisted Mixed-Micelle Formation Results in the Refolding of Unfolded Protein.

The present work reports a new strategy for triblock-copolymer-assisted refolding of sodium dodecyl sulfate (SDS)-induced unfolded serum protein human serum albumin (HSA) by mixed-micelle formation of SDS with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer EO20PO68EO20 (P123) under physiological conditions. The steady-state and time-resolve fluorescence results show that the unfolding of HSA induced by SDS occurs in a stepwise manner through three different phases of binding of SDS, which is followed by a saturation of interaction. Interestingly, the addition of polymeric surfactant P123 to the unfolded protein results in the recovery of ∼87% of its α-helical structure, which was lost during SDS-induced unfolding. This is further corroborated by the return of the steady-state and time-resolved fluorescence decay parameters of the intrinsic tryptophan (Trp214) residue of HSA to the initial nativelike condition. The isothermal titration calorimetry (ITC) data also substantiates that there is almost no interaction between P123 and the native state of the protein. However, the mixed-micelle formation, accompanied by substantial binding affinities, removes the bound SDS molecules from the scaffolds of the unfolded state of the protein. On the basis of our experiments, we conclude that the formation of mixed micelles between SDS and P123 plays a pivotal role in refolding the protein back to its nativelike state.

Contrasting effects of pH on the modulation of the structural integrity of hemoglobin induced by sodium deoxycholate.

Bile salt-mediated conformational modification of hemoglobin (Hb) was examined at three different pHs i.e., 3.2, 7.4 and 9.0. The added bile salt, sodium deoxycholate (NaDC), decreases the α-helicity in Hb (α-helix: 71.3% → 61.7% in the presence of 9.6 mM NaDC, and 83.2% → 66.2% in the presence of 14 mM NaDC, at pH 7.4 and 9.0, respectively), while a reverse pattern of modification in the Circular Dichroism (CD) spectra of Hb is found at pH 3.2. The acid-induced denatured Hb (pH 3.2) regains its structural integrity by changing conformation from a random coil to an α-helix rich secondary structure upon addition of NaDC (α-helix: 10.4% → 53.4%, ß-sheet: 31.0% → 18.5% and random coil: 58.6% → 28.1%, in the presence of 0.65 mM NaDC). Also, a step-wise binding interaction pattern of Hb with NaDC was revealed at pH 7.4 and 9.0 upon variation of steady-state fluorescence intensity and average lifetime of Hb. From the fluorescence lifetime decay pattern, the decrement of energy transfer from Trp to a heme group was found upon the addition of NaDC at pH 7.4 and 9.0. However, at pH 3.2, the modification of the time-resolved fluorescence decay behavior of Hb within NaDC is typically reversed, where the energy transfer from Trp to heme is restored to some extent. Thermodynamic analysis suggests that the Hb-NaDC binding interaction is characterized by a dominant entropic contribution interpreted on the basis of release of ordered water molecules to the bulk aqueous phase.

Hydrophobicity is the governing factor in the interaction of human serum albumin with bile salts.

The present study demonstrates a detailed characterization of the interaction of a series of bile salts, sodium deoxycholate (NaDC), sodium cholate (NaC), and sodium taurocholate (NaTC), with a model transport protein, human serum albumin (HSA). Here, steady-state and time-resolved fluorescence spectroscopic techniques have been used to characterize the interaction of the bile salts with HSA. The binding isotherms constructed from steady-state fluorescence intensity measurements demonstrate that the interaction of the bile salts with HSA can be characterized by three distinct regions, which were also successfully reproduced from the significant variation of the emission wavelength (λ(em)) of the intrinsic tryptophan (Trp) moiety of HSA. The time-resolved fluorescence decay behavior of the Trp residue of HSA was also found to corroborate the steady-state results. The effect of interaction with the bile salts on the native conformation of the protein has been explored in a circular dichroism (CD) study, which reveals a decrease in α-helicity of HSA induced by the bile salts. In accordance with this, the esterase activity of the protein-bile salt aggregates is found to be reduced in comparison to that of the native protein. Our results exclusively highlight the fact that it is the hydrophobic character of the bile salt that governs the extent of interaction with the protein. Isothermal titration calorimetry (ITC) and molecular docking studies further substantiate our other experimental findings.

Inverse Temperature Dependence in Static Quenching versus Calorimetric Exploration: Binding Interaction of Chloramphenicol to ß-Lactoglobulin.

The binding interaction between the whey protein bovine ß-lactoglobulin (ßLG) with the well-known antibiotic chloramphenicol (Clp) is explored by monitoring the intrinsic fluorescence of ßLG. Steady-state and time-resolved fluorescence spectral data reveal that quenching of ßLG fluorescence proceeds through ground state complex formation, i.e., static quenching mechanism. However, the drug-protein binding constant is found to vary proportionately with temperature. This anomalous result is explained on the basis of the Arrhenius theory which states that the rate constant varies proportionally with temperature. Thermodynamic parameters like ΔH, ΔS, ΔG, and the stoichiometry for the binding interaction have been estimated by isothermal titration calorimetric (ITC) study. Thermodynamic data show that the binding phenomenon is mainly an entropy driven process suggesting the major role of hydrophobic interaction forces in the Clp-ßLG binding. Constant pressure heat capacity change (ΔCp) has been calculated from enthalpy of binding at different temperatures which reveals that hydrophobic interaction is the major operating force. The inverse temperature dependence in static quenching is however resolved from ITC data which show that the binding constant regularly decreases with increase in temperature. The modification of native protein conformation due to binding of drug has been monitored by circular dichroism (CD) spectroscopy. The probable binding location of Clp inside ßLG is explored from AutoDock based blind docking simulation.

A critical approach toward resonance-assistance in the intramolecular hydrogen bond interaction of 3,5-diiodosalicylic acid: a spectroscopic and computational investigation.

The photophysics of a prospective drug molecule, 3,5-diiodosalicylic acid (3,5-DISA), having a wide spectrum of biological and medicinal applications, have been investigated using spectroscopic techniques and computational analyses. The remarkably large Stokes' shifts in various solvents from 3,5-DISA has been intertwined with the occurrence of an excited-state intramolecular proton transfer (ESIPT) reaction. Concurrently, the emergence of an intriguing dual emission feature in less interacting solvents is also reported and the spectral response of 3,5-DISA toward the variation of medium acidity/basicity has been exploited to decipher the nature of various species present in different solvents. Our experimental results, unveiling the occurrence of an ESIPT reaction in 3,5-DISA, have been aptly substantiated from computational studies in which the operation of ESIPT has been explored from structural as well as energetics (analysis of potential energy surface (PES)) perspectives. A major focus of the present study is on the evaluation of the intramolecular hydrogen bond (IMHB) interaction in 3,5-DISA, including the application of various methodologies to estimate the IMHB energy and subsequently, an in-depth analysis of the IMHB interaction reveals its partially covalent nature through the application of advanced quantum chemical tools, e.g., the natural bond orbital (NBO) method. In this context, the interplay between the aromaticity of the benzene nucleus and the IMHB energy has been rigorously explored, showing indications for the occurrence of resonance-assisted hydrogen bonding (RAHB) in 3,5-DISA. To this end, the geometric as well as magnetic criteria of aromaticity have been analyzed.

Binding interaction of a prospective chemotherapeutic antibacterial drug with ß-lactoglobulin: results and challenges.

This Article reports a detailed characterization of the binding interaction of a potential chemotherapeutic antibacterial drug, norfloxacin (NOF), with the mammalian milk protein ß-lactoglobulin (ßLG). The thermodynamic parameters, ΔH, ΔS, and ΔG, for the binding phenomenon as-evaluated on the basis of van't Hoff relationship reveal the predominance of electrostatic/ionic interactions underlying the binding process. However, the drug-induced quenching of the intrinsic tryptophanyl fluorescence of the protein exhibits intriguing characteristics on Stern-Volmer analysis (displays an upward curvature instead of conforming to a linear regression). Thus, an extensive time-resolved fluorescence spectroscopic characterization of the quenching process has been undertaken in conjugation with temperature-dependent fluorescence quenching studies to unveil the actual quenching mechanism. The invariance of the fluorescence decay behavior of ßLG as a function of the quencher (here NOF) concentration coupled with the commensurate dependence of the drug-protein binding constant (K) on temperature, the drug-induced fluorescence quenching of ßLG is argued to proceed through static mechanism. This postulate is aided further support from absorption, fluorescence, and circular dichroism (CD) spectral studies. The present study also throws light on the important issue of drug-induced modification in the native protein conformation on the lexicon of CD, excitation-emission matrix spectroscopic techniques. Concurrently, the drug-protein interaction kinetics and the energy of activation of the process are also explored from stopped-flow fluorescence technique. The probable binding locus of NOF in ßLG is investigated from AutoDock-based blind docking simulation.

Binding of norharmane with RNA reveals two thermodynamically different binding modes with opposing heat capacity changes.

The binding interaction of a prospective anti-cancer photosensitizer, norharmane (NHM, 9H-pyrido[3,4-b]indole) with double stranded RNA reveals a primarily intercalative mode of binding. Steady-state and time-resolved fluorescence spectroscopic results demonstrate the occurrence of drug-RNA binding interaction as manifested through environment-sensitive prototropic equilibrium of NHM. However, the key finding of the present study lies in unraveling the complexities in the NHM-RNA binding thermodynamics. Isothermal Titration Calorimetry (ITC) results reveal the presence of two thermodynamically different binding modes for NHM. An extensive temperature-dependence investigation shows that the formation of Complex I is enthalpically (ΔHI < 0) as well as entropically (TΔSI > 0) favored with the enthalpic (entropic) contribution being increasingly predominant in the higher (lower) temperature regime. On the contrary, the formation of Complex II reveals a predominantly enthalpy-driven signature (ΔHI < 0) along with unfavorable entropy change (TΔSI < 0) with gradually decreasing enthalpic contribution with temperature. Such differential dependences of ΔHI and ΔHII on temperature subsequently lead to opposing heat capacity changes underlying the formation of Complex I and II (ΔCpI<0andΔCpII>0). A negative ΔCp underpins the pivotal role of 'hydrophobic effect' (release of ordered water molecules) for the formation of Complex I, while a positive ΔCp marks the thermodynamic hallmark for 'hydrophobic hydration' (solvation of hydrophobic (or nonpolar) molecular surfaces in aqueous medium) for formation of Complex II. A detailed investigation of the effect of ionic strength enables a component analysis of the total free energy change (ΔG).

Association and sequestered dissociation of an anticancer drug from liposome membrane: Role of hydrophobic hydration.

Herein, the interaction of a potent anticancer drug (Sanguinarine, SG) with dimyristoyl-l-α-phosphatidylglycerol (DMPG) liposome membrane has been investigated at physiological pH. The spectroscopic fluorescence decay results demonstrate a modification of the photophysics of SG within DMPG-encapsulated state leading to preferential stabilization of the iminium ion over the alkanolamine form. This suggests a key role of electrostatic force underlying the interaction. The complex dependence of the thermodynamic parameters on temperature yields a unique finding of a positive heat capacity change (ΔCp) indicating the signature of hydrophobic hydration. The study also demonstrates the application of ß-cyclodextrin (ßCD) as a prospective host system resulting in release of the DMPG-bound drug. A calorimetric exploration of the DMPG-ßCD interaction reveals an intrinsically complex thermodynamics of the process leading to ΔCp > 0 and thus marking the instrumental role of hydrophobic hydration which follows that the DMPG-ßCD interaction is accompanied with burial of polar molecular surfaces. A systematic investigation of the diffusion of the drug within various microheterogeneous environments by Fluorescence Correlation Spectroscopy (FCS) categorically reinforces our arguments.

Enhanced Binding of Phenosafranin to Triblock Copolymer F127 Induced by Sodium Dodecyl Sulfate: A Mixed Micellar System as an Efficient Drug Delivery Vehicle.

In this study, we explored the interaction of a cationic phenazinium dye, phenosafranin (PSF, here used as a model drug), with pluronic block copolymer F127, both in the presence and in the absence of the anionic surfactant sodium dodecyl sulfate (SDS), which forms mixed micelles with F127. We applied both steady-state and time-resolved spectroscopic techniques, along with isothermal titration calorimetry (ITC), to demonstrate the binding of the probe PSF to both the pluronic and F127/SDS mixed micelles. Dynamic light scattering (DLS) study revealed that, upon interaction with SDS, the hydrodynamic diameter (dH) of F127 micelles decreased due to the formation of the mixed micelles. The PSF penetrated to the more hydrophobic interior of the mixed micellar system as compared to F127 micelles alone. Micropolarity and fluorescence-quenching experiments revealed that PSF is more deeply seated in the case of F127/SDS mixed micelles. Through a partition coefficient, lifetime measurements, and time-resolved anisotropy experiments, we also established that the partitioning of the probe within the F127 micelles in the presence of SDS is almost double than that in its absence. ITC data corroborates the fact that the binding of PSF is the strongest and most thermodynamically favorable when mixed micelles are formed, which enables our system to serve as an excellent drug delivery vehicle when compared to F127 alone.

Interaction of Bile Salts with ß-Cyclodextrins Reveals Nonclassical Hydrophobic Effect and Enthalpy-Entropy Compensation.

Herein, we present an endeavor toward exploring the lacuna underlying the host:guest chemistry of inclusion complex formation between bile salt(s) and ß-cyclodextrin(s) (ßCDs). An extensive thermodynamic investigation based on isothermal titration calorimetry (ITC) demonstrates a dominant contribution from exothermic enthalpy change (ΔH < 0) accompanying the phenomenon of inclusion complex formation, along with a relatively smaller contribution to total free energy change from the entropic component. However, the negative heat capacity change (ΔCp < 0) displays the hallmark for a pivotal role of hydrophobic effect underlying the interaction. Contrary to the classical hydrophobic effect, such apparently paradoxical thermodynamic signature has been adequately described under the notion of "nonclassical hydrophobic effect". On the basis of our results, the displacement of disordered water from hydrophobic binding sites has been argued to mark the enthalpic signature and the key role of such interaction forces is further corroborated from enthalpy-entropy compensation behavior showing indication for almost complete compensation. To this end, we have quantified the interaction of two bile salt molecules (namely, sodium deoxycholate and sodium glycocholate) with a series of varying chemical substituents on the host counterpart, namely, ßCD, (2-hydroxypropyl)-ßCD, and methyl ßCD.

Interaction of an anti-cancer photosensitizer with a genomic DNA: From base pair specificity and thermodynamic landscape to tuning the rate of detergent-sequestered dissociation.

A detailed characterization of the binding interaction of a potent cancer cell photosensitizer, norharmane (NHM) with a genomic DNA (herring sperm; hsDNA) is undertaken with particular emphasis on deciphering the strength, mode, dynamics, energetics and kinetics of binding. A major focus of the study underlies a successful exploration of the concept of detergent-sequestered dissociation of drug from the drug-DNA complex. Biophysical techniques such as absorption, steady-state and time-resolved fluorescence spectroscopy, circular dichroism, DNA helix melting, stopped-flow fluorescence kinetics and calorimetry have been used. A primarily intercalative mode of binding of NHM with DNA is shown. However, the overall interaction is governed by more than one type of binding forces. We demonstrate that the essential prerequisite of a slower dissociation rate of drug from DNA helix is achieved by tenable choice surfactants. Our results also highlight an effective tunability of the rate of dissociation of the DNA-intercalated drug via detergent-sequestration. A detailed isothermal titration calorimetric study unveils the key role of hydrophobic force underlying NHM-hsDNA association. This is further substantiated by the enthalpy-entropy compensation behavior. The major entropic contribution in detergent-induced dissociation of NHM from NHM-hsDNA complex is also demonstrated. Our results present not only a comprehensive structural and thermodynamic profile, base pair specificity, association kinetics for binding of NHM with DNA but also explore the thermodynamic and kinetic aspects of dissociation of bound drug. Characterization and tuning of the essential prerequisites for a drug to be efficient in anti-cancer functionality bear direct and widespread significance in contemporary global research.

Contrasting Effects of Salt and Temperature on Niosome-Bound Norharmane: Direct Evidence for Positive Heat Capacity Change in the Niosome:ß-Cyclodextrin Interaction.

The modulation of the prototropic equilibrium of a cancer cell photosensitizer, norharmane (NHM), within a niosome microheterogeneous environment has been investigated. The contrasting effects of temperature and extrinsically added salt on the photophysics of niosome-bound drug have been meticulously explored from steady-state and time-resolved spectroscopic techniques. The cation ⇌ neutral prototropic equilibrium of NHM is found to be preferentially favored toward the neutral species with increasing salt concentration, and the results are rationalized on the basis of water penetration to the hydration layer of niosome. The effects are typically reversed with temperature. The differential rotational relaxation behavior of NHM under various conditions has also been addressed from fluorescence anisotropy decay. Further, the study delineates the application of ß-cyclodextrin (ßCD) as a potential host system, leading to drug sequestration from the niosome-encapsulated state. To this end, a detailed investigation of the thermodynamics of the niosome:ßCD interaction has been undertaken by isothermal titration calorimetry (ITC) to unravel the notable dependence of the thermodynamic parameters on temperature. Consequently, a critical analysis of the variation of the enthalpy change (ΔH) of the process with temperature leads to the unique observation of a positive heat capacity change (ΔCp) marking the hallmark of hydrophobic hydration.

Interplay of Multiple Interaction Forces: Binding of Norfloxacin to Human Serum Albumin.

Herein, the binding interaction of a potential chemotherapeutic antibacterial drug norfloxacin (NOF) with a serum transport protein, human serum albumin (HSA), is investigated. The prototropic transformation of the drug (NOF) is found to be remarkably modified following interaction with the protein as manifested through significant modulations of the photophysics of the drug. The predominant zwitterionic form of NOF in aqueous buffer phase undergoes transformation to the cationic form within the protein-encapsulated state. This implies the possible role of electrostatic interaction force in NOF-HSA binding. This postulate is further substantiated from the effect of ionic strength on the interaction process. To this end, the detailed study of the thermodynamics of the drug-protein interaction process from isothermal titration calorimetric (ITC) experiments is found to unfold the signature of electrostatic as well as hydrophobic interaction forces underlying the binding process. Thus, interplay of more than one interaction forces is argued to be responsible for the overall drug-protein binding. The ITC results reveal an important finding in terms of enthalpy-entropy compensation (EEC) characterizing the NOF-HSA binding. The effect of drug-binding on the native protein conformation has also been evaluated from circular dichroism (CD) spectroscopy which unveils partial rupture of the protein secondary structure. In conjunction to this, the functionality of the native protein (in terms of esterase-like activity) is found to be lowered as a result of binding with NOF. The AutoDock-based docking simulation unravels the probable binding location of NOF within the hydrophilic subdomain IA of HSA. The present program also focuses on exploring the dynamical aspects of the drug-protein interaction scenario. The rotational-relaxation dynamics of the protein-bound drug reveals the not-so-common "dip-and-rise" pattern.

Prototropic transformation and rotational-relaxation dynamics of a biological photosensitizer norharmane inside nonionic micellar aggregates.

The effect of variation of the size of headgroup as well as the length of hydrocarbon tail of nonionic surfactants on the photophysics and rotational-relaxation dynamics of a promising biological photosensitizer, norharmane, (NHM) has been investigated. The series of nonionic micelles employed for the study belongs to Triton X family (allowing the variation in poly(ethylene oxide) (PEO) chain length) and Tween family (providing access to vary the alkyl chain length of the surfactant tails). The spectral deciphering of the photophysics of the drug (NHM) within the series of the nonionic micelles reveals remarkable influence of binding of the drug with the micelles on the prototropic equilibrium which is notably favored toward the neutral species of the drug over the cationic counterpart. The strength of drug-micelle binding interaction as well as the extent of transformation of the cation ⇌ neutral prototropic equilibrium is found to be enormously governed by the variation of the headgroup size and the alkyl chain length of the surfactants. To this end, the equilibrium constant (Keq) and free energy change (ΔG) for cation ⇌ neutral prototropic transformation of the drug as a function of the micellar parameters have been meticulously explored from emission studies and comprehensively rationalized under the provision of the micellar hydration model, that is, the differential extents of water penetration to micellar units as a function of varying thickness of the palisade layer and hence a variation in the polarity of the micellar microenvironments. The significant enhancement in steady-state fluorescence anisotropy of NHM in micellar environments compared to that in bulk aqueous buffer phase substantiates the location of the drug in motionally constrained regions introduced by the nonionic micelles. Further, all these lines of arguments are effectively corroborated from time-resolved fluorescence experiments with particular emphasis on time-resolved anisotropy decay study of the drug within the micellar aggregates.

Temperature induced morphological transitions from native to unfolded aggregated States of human serum albumin.

The circulatory protein, human serum albumin (HSA), is known to have two melting point temperatures, 56 and 62 °C. In this present manuscript, we investigate the interaction of HSA with a synthesized bioactive molecule 3-pyrazolyl 2-pyrazoline (PZ). The sole tryptophan amino acid residue (Trp214) of HSA and PZ forms an excellent FRET pair and has been used to monitor the conformational dynamics in HSA as a function of temperature. Molecular docking studies reveal that the PZ binds to a site which is in the immediate vicinity of Trp214, and such data are also supported by time-resolved FRET studies. Steady-state and time-resolved anisotropy of PZ conclusively proved that the structural and morphological changes in HSA mainly occur beyond its first melting temperature. Although the protein undergoes thermal denaturation at elevated temperatures, the Trp214 gets buried inside the protein scaffolds; this fact has been substantiated by acrylamide quenching studies. Finally, we have used atomic force microscopy to establish that at around 70 °C, HSA undergoes self-assembly to form fibrillar structures. Such an observation may be attributed to the loss of α-helical content of the protein and a subsequent rise in ß-sheet structure.