Domain-Specific Association of a Phenanthrene-Pyrene-Based Synthetic Fluorescent Probe with Bovine Serum Albumin: Spectroscopic and Molecular Docking Analysis.
ABSTRACT: In this report, the interaction between a phenanthrene-pyrene-based fluorescent probe (PPI) and bovine serum albumin (BSA), a transport protein, has been explored by steady-state emission spectroscopy, fluorescence anisotropy, far-ultraviolet circular dichroism (CD), time-resolved spectral measurements, and molecular docking simulation study. The blue shift along with emission enhancement indicates the interaction between PPI and BSA. The binding of the probe causes quenching of BSA fluorescence through both static and dynamic quenching mechanisms, revealing a 1:1 interaction, as delineated from Benesi-Hildebrand plot, with a binding constant of ?105 M-1, which is in excellent agreement with the binding constant extracted from fluorescence anisotropy measurements. The thermodynamic parameters, ?H°, ?S°, and ?G°, as determined from van't Hoff relationship indicate the predominance of van der Waals/extensive hydrogen-bonding interactions for the binding phenomenon. The molecular docking and site-selective binding studies reveal the predominant binding of PPI in subdomain IIA of BSA. From the fluorescence resonance energy transfer study, the average distance between tryptophan 213 of the BSA donor and the PPI acceptor is found to be 3.04 nm. CD study demonstrates the reduction of ?-helical content of BSA protein on binding with PPI, clearly indicating the change of conformation of BSA.
Project description:Steady state and time resolved fluorescence along with anisotropy and induced circular dichroism (ICD) spectroscopy provide useful tools to observe and understand the behavior of the therapeutically important plant flavonoids fisetin and daidzein in ?-cyclodextrin (?-CDx) nanocavity. Benesi-Hildebrand plots indicated 1:1 stoichiometry for both the supramolecular complexes. However, the mode of the binding of fisetin significantly differs from daidzein in ?-CDx, as is observed from ICD spectra which is further confirmed by docking studies. The interaction with ?-CDx proceeds mainly by the phenyl ring and partly by the chromone ring of fisetin whereas only the phenyl ring takes part for daidzein. A linear increase in the aqueous solubility of the flavonoids is assessed from the increase in the binding of the flavonoids with the ?-CDx cavity, which are determined by the gradual increase in the ICD signal, fluorescence emission as well as increase in fluorescence anisotropy with increasing (?-CDx). This confirms ?-CDx as a nanovehicle for the flavonoids fisetin and daidzein in improving their bioavailability.
Project description:This study investigated the interaction between eupatorin and bovine serum albumin (BSA) using ultraviolet-visible (UV-vis) absorption, fluorescence, synchronous fluorescence, circular dichroism (CD) spectroscopies, and molecular modeling at pH 7.4. Results of UV-vis and fluorescence spectroscopies illustrated that BSA fluorescence was quenched by eupatorin via a static quenching mechanism. Thermodynamic parameters revealed that hydrophobic and electrostatic interactions played major roles in the interaction. Moreover, the efficiency of energy transfer, and the distance between BSA and acceptor eupatorin, were calculated. The effects of eupatorin on the BSA conformation were analyzed using UV-vis, CD, and synchronous fluorescence. Finally, the binding of eupatorin to BSA was modeled using the molecular docking method.
Project description:Triptolide is a major active constituent isolated from Tripterygiumwilfordii Hook F, a Chinese herbal medicine. This study investigated the intermolecular interaction between triptolide and bovine serum albumin (BSA).The fluorescence, circular dichroism (CD) and molecular docking methods were used to investigate the intermolecular interaction between triptolide and BSA. The binding constant, the number of binding sites, binding subdomain and the thermodynamic parameters were measured.The results of this experiment revealed that the intrinsic fluorescence of BSA was effectively quenched by triptolide via static quenching. The experimental results of synchronous fluorescence and CD spectra showed that the conformation of BSA was changed in the presence of triptolide.It indicated that triptolide could spontaneously bind on site II (subdomain IIIA) of BSA mainly via hydrogen bonding interactions and Van der Waals force.
Project description:Sodium benzoate (SB) is widely used as a preservative in food industry, and bovine serum albumin (BSA) is a major carrier protein similar to human serum albumin (HSA), the study of the binding between the two has great significance on human health. In this paper, we systematically investigated the binding of SB and BSA under the simulated physiological conditions combining with various common analytical methods, e.g., fluorescence, UV-vis absorption, synchronous fluorescence and circular dichroism (CD) spectra, as well as molecular docking method. The fluorescence quenching measurements were respectively carried out at 298 K, 303 K and 308 K using the Stern-Volmer method. The results reveal that ground state SB-BSA complex was formed within the binding constants from 2.02?×?104 to 7.9?×?103 M-1. Meanwhile, the negative values of ?H 0 (-?43.92 kJ mol-1) and ?S 0 (-?111.6 J mol-1 K-1) demonstrated that both the hydrogen binding interaction and van der Waals forces contributed to stabilizing the SB-BSA complex. The site marker competitive experiments show that the SB and BSA bound at site I. Furthermore, the experimental results of UV-vis absorption, synchronous fluorescence and CD spectra indicate that the binding of SB and BSA may change the conformation of BSA. In addition, the molecular docking experiment suggests that hydrogen bond was formed in the interaction between SB and BSA.
Project description:Interaction between bovine serum albumin (BSA) and phosphorus heterocycles (PHs) was studied using multi-spectroscopic techniques. The results indicated the high binding affinity of PHs to BSA as it quenches the intrinsic fluorescence of BSA. The experimental data suggested the fluorescence quenching mechanism between PHs and BSA as a dynamic quenching. From the UV-vis studies, the apparent association constant (Kapp) was found to be 9.25×102, 1.27×104 and 9.01×102 L/mol for the interaction of BSA with PH-1, PH-2 and PH-3 respectively. According to the Förster's non-radiation energy transfer (FRET) theory, the binding distances between BSA and PHs were calculated. The binding distances (r) of PH-1, PH-2 and PH-3 were found to be 2.86, 3.03, and 5.12 nm, respectively, indicating energy transfer occurs between BSA and PHs. The binding constants of the PHs obtained from the fluorescence quenching data were found to be decreased with increase of temperature. The negative values of the thermodynamic parameters ?H, ?S and ?G at different temperatures revealed that the binding process is spontaneous; hydrogen bonds and van der Waals interaction were the main force to stabilize the complex. The microenvironment of the protein-binding site was studied by synchronous fluorescence and circular dichroism (CD) techniques and data indicated that the conformation of BSA changed in the presence of PHs. Finally, we studied the BSA-PHs docking using Autodock and results suggest that PHs is located in the cleft between the domains of BSA.
Project description:Interaction mechanisms of human serum albumin (HSA) with safranal and crocin were studied using UV-Vis absorption, fluorescence quenching and circular dichroism (CD) spectroscopies as well as molecular docking techniques. Changes in absorbance and fluorescence of HSA upon interactions with both compounds were attributed to their binding to amino acid chromophores located in subdomains IIA and IIIA. Fluorescence secondary inner filter effect was excluded using 278?nm and 340?nm as the wavelengths of HSA's excitation and fluorescence while safranal and crocin absorbed at 320?nm and 445?nm, respectively. Stern-Volmer model revealed a static quenching mechanism involve the formation of non-fluorescent ground state complexes. Stern-Volmer, Hill, Benesi-Hilbrand and Scatchard models gave apparent binding constants ranged in 4.25?×?103 - 2.15?×?105 for safranal and 7.67?×?103 - 4.23?×?105?L mol-1 for crocin. CD measurements indicated that 13 folds of safranal and crocin unfolded the ?-helix structure of HSA by 7.47-21.20%. In-silico molecular docking revealed selective exothermic binding of safranal on eight binding sites with binding energies ranged in -3.969 to -6.6.913?kcal/mol. Crocin exothermally bound to a new large pocket located on subdomain IIA (sudlow 1) with binding energy of -12.922?kcal/mol. These results confirmed the formation of HSA stable complexes with safranal and crocin and contributed to our understanding for their binding characteristics (affinities, sites, modes, forces … etc.) and structural changes upon interactions. They also proved that HSA can solubilize and transport both compounds in blood to target tissues. The results are of high importance in determining the pharmacological properties of the two phytochemical compounds and for their future developments as anticancer, antispasmodic, antidepressant or aphrodisiac therapeutic agents.
Project description:The binding interaction between bovine serum albumin (BSA) and sodium salt of risedronic acid (RSN) was studied by using the FT-IR (Fourier transform infrared), UV-Vis (ultraviolet-visible), fluorescence (emission and synchronous), CD (circular dichroism) spectrometric, and computational (molecular docking) techniques at 289, 297, and 305?K temperatures with physiological buffer of pH 7.40. The conformational and secondary structural changes observed for BSA from CD spectra and by curve fitting procedure were applied to Fourier self-deconvolution in FT-IR spectra. The formation of a BSA-RSN complex was confirmed from UV-Vis spectroscopy. The static type of quenching shown for RSN to BSA was verified from Stern-Volmer and modified Stern-Volmer equations. The binding constant of order 105 was obtained to be confirming that there exists a strong binding interaction between BSA and RSN. Synchronous fluorescence shows that the microenvironment of tryptophan was altered, not tyrosine of BSA; in addition to this, the distance between tryptophan of BSA and RSN was found out from Forster's theory of nonradiation energy transfer. The interaction between BSA and RSN mainly occurred as a result of hydrogen bonds and van der Waals forces, the process is exothermic and spontaneous, and it was achieved through van 't Hoff equation. This interaction was affected by the presence of biologically active Fe2+, Ni2+, Ca2+, Mg2+, and Cd2+ ions and was also studied. The subdomain IIIA of BSA involved with RSN interaction was authenticated from molecular docking analysis.
Project description:Phytochemical investigation on the methanol extract of Woodwardia unigemmata resulted in the isolation of seven flavonoids, including one new flavonol acylglycoside (1). The structures of these compounds were elucidated on the basis of extensive spectroscopic analysis and comparison of literature data. The multidrug resistance (MDR) reversing activity was evaluated for the isolated compounds using doxorubicin-resistant K562/A02 cells model. Compound 6 showed comparable MDR reversing effect to verapamil. Furthermore, the interaction between compounds and bovine serum albumin (BSA) was investigated by spectroscopic methods, including steady-state fluorescence, synchronous fluorescence, circular dichroism (CD) spectroscopies, and molecular docking approach. The experimental results indicated that the seven flavonoids bind to BSA by static quenching mechanisms. The negative ?H and ?S values indicated that van der Waals interactions and hydrogen bonds contributed in the binding of compounds 2-6 to BSA. In the case of compounds 1 and 7 systems, the hydrophobic interactions play a major role. The binding of compounds to BSA causes slight changes in the secondary structure of BSA. There are two binding sites of compound 6 on BSA and site I is the main site according to the molecular docking studies and the site marker competitive binding assay.
Project description:Recognition of elements of protein tertiary structure is crucial for biotechnological and biomedical tasks; this makes the development of optical sensors for certain protein surface elements important. Herein, we demonstrated the ability of iron(II) clathrochelates (<b>1</b>-<b>3</b>) functionalized with mono-, di- and hexa-carboxyalkylsulfide to induce selective circular dichroism (CD) response upon binding to globular proteins. Thus, inherently CD-silent clathrochelates revealed selective inducing of CD spectra when binding to human serum albumin (HSA) (<b>1</b>, <b>2</b>), beta-lactoglobuline (<b>2</b>) and bovine serum albumin (BSA) (<b>3</b>). Hence, functionalization of iron(II) clathrochelates with the carboxyalkylsulfide group appears to be a promising tool for the design of CD-probes sensitive to certain surface elements of proteins tertiary structure. Additionally, interaction of <b>1</b>-<b>3</b> with proteins was also studied by isothermal titration calorimetry, protein fluorescence quenching, electrospray ionization mass spectrometry (ESI-MS) and computer simulations. Formation of both 1:1 and 1:2 assemblies of HSA with <b>1</b>-<b>3</b> was evidenced by ESI-MS. A protein fluorescence quenching study suggests that <b>3</b> binds with both BSA and HSA via the sites close to Trp residues. Molecular docking calculations indicate that for both BSA and HSA, binding of <b>3</b> to Site I and to an "additional site" is more favorable energetically than binding to Site II.
Project description:The drug, di-2-pyridylketone-2-pyridine carboxylic acid hydrazone (DPPCAH) and its copper complex (DPPCAH-Cu) exhibit significant antitumor activity. However, the mechanism of their pharmacological interaction with the biological molecule bovine serum albumin (BSA) remains poorly understood. The present study elucidates the interactions between the drug and BSA through MTT assays, spectroscopic methods and molecular docking analysis. Our results indicate that BSA could attenuate effect on the cytotoxicity of DPPCAH, but not DPPCAH-Cu. Data from fluorescence quenching measurements demonstrated that both DPPCAH and DPPCAH-Cu could bind to BSA, with a reversed effect on the environment of tryptophan residues in polarity. CD spectra revealed that the DPPCAH-Cu exerted a slightly stronger effect on the secondary structure of BSA than DPPCAH. The association constant of DPPCAH with BSA was greater than that of DPPCAH-Cu. Docking studies indicated that the binding of DPPCAH to BSA involved a greater number of hydrogen bonds compared to DPPCAH-Cu. The calculated distances between bound ligands and tryptophans in BSA were in agreement with fluorescence resonance energy transfer results. Thus, the binding affinity of the drug (DPPCAH or DPPCAH-Cu) with BSA partially contributes to its antitumor activity; the greater the drug affinity is to BSA, the less is its antitumor activity.