Modulation of Prototropic Activity and Rotational Relaxation Dynamics of a Cationic Biological Photosensitizer within the Motionally Constrained Bio-environment of a Protein (original) (raw)
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Physical Chemistry Chemical Physics, 2012
The present work demonstrates the effect of biological confinement on the photophysics and dynamics of a bio-active drug molecule viz., 5-chlorosalicylic acid (5ClSA). 5ClSA is a potential candidate exhibiting Excited-State Intramolecular Proton Transfer (ESIPT) reaction and thereby generating the phototautomer (i.e. proton transferred keto form) in the excited state. Given the pK a of 5ClSA (around 2.64), the anionic form of the drug molecule is expected to be the interacting species with the protein under the experimental conditions (buffered solution of pH 7.40). The ESIPT photophysics of the drug (5ClSA anion) is found to be remarkably modified within the confined bio-environment of a model transport protein Bovine Serum Albumin (BSA) in terms of remarkable emission intensity enhancement coupled with a discernible red-shift of the emission maximum wavelength. Such considerable modification of the ESIPT photophysics of the 5ClSA anion has been exploited to determine the drug-protein binding strength (as characterized by the binding constant K (AE10%) = 6.11 Â 10 2 M À1). The present work also delves into evaluation of the probable binding location of the drug within the biomacromolecular assembly of the protein by a blind docking simulation technique, which reveals hydrophobic subdomain IIA to be the probable binding site of the drug. Circular dichroism (CD) spectroscopy delineates the effect of drug binding on the protein secondary structure in terms of decrease of a-helical content of BSA with increasing drug concentration. Apart from this, the excitation-emission matrix fluorescence technique is found to hint at the effect on protein tertiary structure upon binding to the drug. Chaotrope-induced protein denaturation has been explored to complement the findings on the binding interaction process. The modulated dynamics of the proton transfer phototautomer of the 5ClSA anion within the biological confinement is also investigated in this context to explore the slower rate of solvent-relaxation dynamics.
Physical Chemistry Chemical Physics, 2005
A combined approach using global analysis of circular dichroism multiwavelength data and time resolved fluorescence was applied to investigate the interaction of R-(À)-and S-(þ)-ketoprofen with bovine serum albumin in buffer solution at neutral pH. A characterization of the most stable drug : protein adducts of 1 : 1 and 2 : 1 stoichiometry, as individual chemical species, was obtained. The stability constants and the absolute circular dichroism spectra of the diastereomeric complexes were determined. The spectra of the 1 : 1 conjugates are opposite in sign, those of the 2 : 1 complexes are both negative, but different in shape from each other (peaks at 358 and 342 nm for S-(þ)-and R-(À)-ketoprofen, respectively). A tryptophan residue was shown to be involved in the binding of the drug, in the primary site for the R-(À) and in the secondary site for the S-(þ) enantiomer, thereby showing that chiral recognition by the protein causes the site of highest affinity being not the same for both optical antipodes.
Journal of Photochemistry and Photobiology B: Biology, 2013
The present contribution reports a detailed characterization of the binding interaction of two potential anticancer, anti-HIV drugs isatin (IST) and 1-methylisatin (MI) with model transport protein Bovine Serum Albumin (BSA). Thermodynamic parameters e.g., DH, DS and DG for the binding phenomenon have been evaluated on the basis of van't Hoff equation to understand the force behind the binding process. A combined application of steady-state and time-resolved fluorescence spectroscopic techniques substantiate the observed drug-induced quenching of intrinsic tryptophanyl fluorescence of the protein to proceed through a static mechanism. Circular dichroic (CD), synchronous fluorescence and excitationemission matrix fluorescence spectroscopic techniques have been exploited to delineate the secondary and tertiary conformational changes in the protein structure induced by the binding of drugs (IST/MI). The probable binding location of the drug molecules within the protein cavity (hydrophobic subdomain IIIA) has been explored from AutoDock-based blind docking simulation. Examination of drug-protein binding kinetics using stopped-flow fluorescence technique reveals that the association constants (k a) for IST-BSA and MI-BSA interactions are 1.09 Â 10 À3 s À1 (±5%) and 1.73 Â 10 À3 s À1 (±5%), respectively, at the experimental temperature (T) of 298 K. The present study also delves into the effect of drug-binding on the esterase activity of the protein which is found to be reduced in the drug-protein conjugate system in comparison with the native protein.
BioNanoScience, 2019
Benzocaine drug (BZC) is an active component of various nonprescription drugs and used for numb teething treatments. The interaction of BZC with bovine serum albumin (BSA) has been studied using fluorescence, synchronous fluorescence, UV-Vis, circular dichroism (CD), and molecular docking analysis. The results revealed that BZC has a strong affinity to quench the intrinsic fluorescence of BSA in terms of a static quenching mechanism under physiological conditions. The fluorescence quenching data revealed that the quenching constants are (K SV) 4.10, 3.30, and 2.35 × 10 4 L mol −1 at 298, 304, and 310 K, respectively. The binding constants (K b) at three different temperatures (298, 304, and 310 K) were found to be 6.02, 3.72, and 1.10 × 10 5 L mol −1 , respectively. The thermodynamic parameters ΔH°and ΔS°have been estimated to be − 70.67 and − 128.9 J mol −1 K −1 , respectively, thereby, indicating that hydrogen bonding and Van der Waals forces play major role in the interaction of BSA-BZC. Moreover, the negative values of ΔG°− 32.30, − 31.50, − 30.68 kJ mol −1 at 298, 304, 310 K, respectively, indicate the spontaneity of the interaction. FRET analysis proved high probability of energy transfer from BSA to the drug molecule. Molecular docking and displacement studies indicated that BZC was bound to the Sudlow's site II through hydrogen bonding and Van der Waals interactions.
Binding Interaction of a Biological Photosensitizer with Serum Albumins: A Biophysical Study
Biomacromolecules, 2007
A photophysical study on the binding interaction of an efficient cancer cell photosensitizer, norharmane (NHM), with model transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), has been performed using a combination of steady-state and time-resolved fluorescence techniques. The emission profile undergoes a remarkable change upon addition of the proteins to the buffered aqueous solution of the photosensitizer. The polarity-dependent prototropic transformation is responsible for the remarkable sensitivity of this biological fluorophore to the protein environments. A marked increase in the fluorescence anisotropy in the proteinous environments indicates that the albumin proteins introduce motional restriction on the drug molecule. Light has been thrown on the denaturing action of urea on the probe-bound protein. The probable binding site of the drug in proteins has also been assessed from the combination of denaturation study, micropolarity measurement, and fluorescence resonance energy transfer (FRET) study. The present study suggests that the stability of serum albumins is enhanced upon binding with the drug.
The present study describes the insights in to the mechanism of interaction between a model protein and an anticancer drug, dasatinib (DAS). DAS is a multi-target anticancer drug which controls the proliferation of mutant expressing cells. Human serum albumin (HSA) is the ubiquitous protein of blood plasma (40-50 g/L) and able to bind a wide range of exogenous and endogenous ligands. Binding mechanism of DAS to HSA was studied under physiological conditions by multi-spectroscopic techniques viz., steady state fluorescence, time resolved fluorescence, fluorescence (Fӧrster) resonance energy transfer (FRET), UV-visible and FTIR besides molecular modeling studies. Fluorescence measurements showed that the fluorescence intensity of HSA was quenched significantly by DAS with a red shift (from 342 to 356 nm) in its emission wavelength. The values of binding constant, number of binding sites and Stern-Volmer quenching constant (K sv) were calculated for DAS-HSA interactions at different temperatures. Decreased K sv values with increase in temperature suggested the presence of static quenching mechanism. This was also evident from time resolved fluorescence studies. The probable binding site for DAS in the hydrophobic pocket (sub-domain IIA) of HSA was revealed by both site probe experiments and molecular docking studies. Negative values of ΔG°revealed the spontaneity of DAS-HSA interaction while negative ΔH°and ΔS°values indicated the participation of hydrogen bonding and van der Waals forces in the stabilization of DAS-HSA complex. Further, conformational change in the protein backbone upon the addition of DAS was evident from absorption, FT-IR and synchronous fluorescence spectral results. The effect of essential metal ions on the binding of DAS to protein was also examined.
The Journal of Physical Chemistry B, 2009
Interaction of bovine serum albumin (BSA) with two series of dipolar molecules having both rigid and flexible structures has been studied by monitoring the spectral and temporal behavior of the intramolecular charge transfer fluorescence of the systems. The binding sites of the molecular systems in BSA have been located with the help of docking studies. Three different sites of varying hydrophobicity have been identified where these molecules are located. Binding in the hydrophobic domains of BSA leads to a blue shift of the fluorescence spectra and an enhancement of fluorescence intensity and lifetime. This enhancement is found to be the largest for flexible systems in which internal motion serves as a nonradiative decay route. In the BSA-bound condition, some of the dipolar molecules exhibit not-so-common "dip-rise-dip" time-resolved fluorescence anisotropy profiles. It is shown that a large difference of the fluorescence lifetimes of the protein-bound and unbound molecules is one of the factors that contributes to this kind of anisotropy profiles. As internal motion is often responsible for the short fluorescence lifetime of the flexible dipolar molecules, a large increase in the fluorescence lifetime of these systems occurs if binding to BSA leads to disruption/prevention of this motion. It thus appears that it might be possible to obtain information on the prevention/disruption of nonradiative pathway on protein binding from the anisotropy profiles of the kind discussed above. However, since the present study reveals cases where a large change in fluorescence lifetime also occurs due to other reasons, one needs to be careful prior to making any conclusion.
Probing Protein Binding Sites by Circular Dichroism Spectroscopy
Current Drug Discovery Technologies, 2004
Pharmacological and pharmacodynamic properties of biologically active natural and synthetic compounds are crucially determined via their binding to proteins of the human body. Several spectroscopic techniques are available to study these mainly non-covalent interactions. Circular dichroism (CD) spectroscopy, being sensitive to the chirality of ligand molecules induced by the asymmetric protein environment, has widely and successfully been applied for many decades. Chiral conformation of the ligand due to conformational adaptation to its binding site, or interaction between ligand molecules held in chiral arrangement relative to each other by the protein sites, results in one or more induced CD bands with different shape, sign and intensity. These extrinsic Cotton effects present in light absorbing region of the optically active or inactive ligand molecules give qualitative and quantitative information of the binding process. It can provide valuable data on the stereochemistry, number, location and nature of the binding sites. This paper is aimed to survey briefly the literature and the results of recent investigations undertaken in this field.