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Binding interaction of a novel fluorophore with serum albumins: steady state fluorescence perturbation and molecular modeling analysis.

ABSTRACT: Fluorescence emission and anisotropy are widely used to measure the binding parameters and kinetic behavior of reactions that cause a change in the rotational time of a fluorescent molecule. We report here fluorescence emission and anisotropy behavior of a newly synthesized novel naphthalene base fluorophore (methyl 3-[(6-{[2-(tert-butoxy)-2-oxoethyl] (4-methoxyphenyl)amino}naphthalen-2-yl)formamido]propanoate) in several solution conditions including its binding to human and bovine serum albumin proteins both in their native and denatured states. The fluorescence yield of the compound substantially increased inside hydrophobic protein surface and ~30 nm decrease in Stokes' shift, compared to aqueous solution, was observed. Shift in fluorescence excitation peak position from the absorption peak of the molecule was ~8 nm in protein solution. This indicated possible alteration of excited state geometry of the compound by the globular fold of albumins. In addition, we measured the steady state fluorescence anisotropy of the molecule to evaluate several thermodynamic parameters and the results suggested the binding was energetically favorable. The measured ?G° was ~-30 kJ mol(-1) and the derived dissociation constant was ~10(-6) M. The molecular docking analysis further highlighted the nonspecific association of the compound with the proteins and hydrophobic forces may have a significant role in the binding processes. Under the denatured condition of the protein, the compound lost its binding efficacy and reduction in fluorescence intensity was observed. Thus, the molecule appears as a new fluorescence probe to report the nature of its binding site in terms of increased fluorescence quantum yield and decreased Stokes' shift. It can also report the changes in the binding site due to global change in protein structure such as unfolding/misfolding often linked to several human disorder. Further it could be useful to detect and study the drug binding site of specific protein of interest.

SUBMITTER: Pal U

PROVIDER: S-EPMC4582037 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Binding interaction of a novel fluorophore with serum albumins: steady state fluorescence perturbation and molecular modeling analysis.

Pal Uttam U Pramanik Sumit Kumar SK Bhattacharya Baisali B Banerji Biswadip B Maiti Nakul Chandra NC

SpringerPlus 20150924

Fluorescence emission and anisotropy are widely used to measure the binding parameters and kinetic behavior of reactions that cause a change in the rotational time of a fluorescent molecule. We report here fluorescence emission and anisotropy behavior of a newly synthesized novel naphthalene base fluorophore (methyl 3-[(6-{[2-(tert-butoxy)-2-oxoethyl] (4-methoxyphenyl)amino}naphthalen-2-yl)formamido]propanoate) in several solution conditions including its binding to human and bovine serum albumi ...[more]

PMID: 26435894

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Project description:The â??small perturbationâ?? approach is critical in studying the â??steady stateâ?? of a biological system. In our experiments, small perturbations were generated by applying a series of repeating intermittent small doses of ultraviolet radiation to a human keratinocyte cell line, HaCaT. The biological response was assessed by monitoring the gene expression profiles using a high reliability and high resolution cDNA microarray system. Following intermittent 10 J/m2 UVB small perturbations, two opposite classes of genes, down-regulated and up-regulated, exhibited an immediate response followed by relaxation between each small perturbation, but were prolonged down- or up-regulated without relaxation while larger doses (233 or 582.5 J/m2) of UVB were applied. A repeated cycle pattern of gene expression following small perturbations is an indication of the existence of steady states. This cycle pattern is suppressed when large perturbations are applied. We believe that this is a universal phenomenon. In our experiments, the functions of up-regulated genes were mainly associated with anti-proliferation, anti-mitogenesis, and apoptosis. On the other hand, down-regulated genes were mainly related to proliferation, mitogenesis, and anti-apoptosis. In conclusion, this study experimentally proves the concept of steady state at the transcription level and demonstrates the feasibility of using small perturbation approaches for investigating steady states. This study could also set a foundation of computational systems biology, which has implicitly used the concept of steady state. Keywords: time course Three UVB exposures are indicated by UV at time points of 0, 8 and 16 hours. T1, T2, T3, T4, T5, and T6 denote the sampling time points. T1, T3 and T5 are allocated 30 minutes after the corresponding UVB irradiation. T2, T4 and T6 are allocated 8 hours after each UVB irradiation. At each sampling time point, two samples (control and UV-irradiated) are collected. See supplementary file Loop_design.pdf for further explanation.

Project description:gamma-Aminobutyric acid (GABA) is a naturally occurring inhibitory neurotransmitter and some of its derivatives showed potential to act as neuroprotective agents. With the aim of developing potential leads for anti-Alzheimer's drugs, in this study we synthesized a novel GABA derivative, methyl 4-(4-((2-(tert-butoxy)-2-oxoethyl)(4-methoxyphenyl)amino)benzamido)butanoate by a unique method of Buchwald-Hartwig cross coupling synthesis; with some modification the yield was significant (97 %) and spectroscopic analysis confirmed that the compound was highly pure (98.8 % by HPLC). The druglikeness properties such as logP, logS, and polar surface area were 3.87, -4.86 and 94.17 Å(2) respectively and it satisfied the Lipinski's rule of five. We examined the binding behavior of the molecule to human serum albumin (HSA) and bovine serum albumin (BSA) which are known as universal drug carrier proteins. The molecule binds to the proteins with low micromolar efficiency and the calculated binding constants were 3.85 and 2.75 micromolar for BSA and HSA, respectively. Temperature dependent study using van't Hoff equation established that the binding was thermodynamically favorable and the changes in the Gibb's free energy, ?G for the binding process was negative. However, the binding of the molecule to HSA was enthalpy driven and the change of enthalpy (?H) was -10.63 kJ/mol, whereas, the binding to BSA was entropy driven and the change in entropy ?S was 222 J/mol. The molecular docking analysis showed that the binding sites of the molecule lie in the groove between domain I and domain III of BSA, whereas it is within the domain I in case of HSA, which also supported the different thermodynamic nature of binding with HSA and BSA. Molecular dynamics analysis suggested that the binding was stable with time and provided further details of the binding interaction. Molecular dynamics study also highlighted the effect of this ligand binding on the serum albumin structure.

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Binding interaction of a novel fluorophore with serum albumins: steady state fluorescence perturbation and molecular modeling analysis.

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Binding interaction of a novel fluorophore with serum albumins: steady state fluorescence perturbation and molecular modeling analysis.

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