Spectroscopic analysis on the interaction of ferulic acid and tetramethylpyrazine with trypsin

The interaction of trypsin with tetramethylpyrazine (TMP) and ferulic acid (FA) was studied using fluorescence, synchronous fluorescence, UV–vis absorption, circular dichroism (CD) and three‐dimensional (3D) fluorescence spectra techniques. Using fluorescence quenching calculations, the bimolecular quenching constant (k_q), apparent quenching constant (K_SV), effective binding constant (K_a) and binding site number (n) were obtained. The distance r between donor and acceptor was found to be 2.049 and 1.281 nm for TMP–trypsin and FA–trypsin complexes. TMP and FA can quench the fluorescence intensity of trypsin by a static quenching procedure. Thermodynamic parameters calculated on the basis of different temperatures revealed that the binding of trypsin to TMP/FA mainly depended on van der Waals' forces and hydrogen bonds. The effect of TMP and FA on the conformation of trypsin was analyzed using synchronous fluorescence, CD, 3D fluorescence spectra and molecular docking studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparison and analysis on the serum‐binding characteristics of aspirin–zinc complex and aspirin

This study was designed to compare the protein‐binding characteristics of aspirin–zinc complex (AZN) with those of aspirin itself. AZN was synthesized and interacted with a model transport protein, human serum albumin (HSA). Three‐dimensional fluorescence, ultraviolet–visible and circular dichroism (CD) spectra were used to characterize the interaction of AZN with HSA under physiological conditions. The interaction mechanism was explored using a fluorescence quenching method and thermodynamic calculation. The binding site and binding locality of AZN on HSA were demonstrated using a fluorescence probe technique and Förster non‐radiation energy transfer theory. Synchronous fluorescence and CD spectra were employed to reveal the effect of AZN on the native conformation of the protein. The HSA‐binding results for AZN were compared with those for aspirin under consistent experimental conditions, and indicated that aspirin acts as a guide in AZN when binding to Sudlow's site I, in subdomain IIA of the HSA molecule. Moreover, compared with aspirin, AZN showed greater observed binding constants with, but smaller changes in the α‐helicity of, HSA, which proved that AZN might be easier to transport and have less toxicity in vivo.     

Characterization of colchicine binding with normal and glycated albumin: In vitro and molecular docking analysis

The transport of more than 90% of the drugs viz. anticoagulants, analgesics, and general anesthetics in the blood takes place by albumin. Hence, albumin is the prime protein needs to be investigated to find out the nature of drug binding. Serum albumin molecules are prone to glycation at elevated blood glucose levels as observed in diabetics. In this piece of work, glycation of bovine serum albumin (BSA) was carried out with glyceraldehyde and characterized by molecular docking and fluorometry techniques. Glycation of BSA showed 25% loss of free amino groups and decreased protein fluorescence (60%) with blue shift of 6 nm. The present study was also designed to evaluate the binding of colchicine (an anti-inflammatory drug) to native and glycated BSA and its ability to displace 8-analino-1-nephthalene sulfonic acid (ANS), from the BSA–ANS complex. Binding of ANS to BSA showed strong binding (K_a = 4.4 μM) with native conformation in comparison to glycated state (K_a = 8.4 μM). On the other hand, colchicine was able to quench the fluorescence of native BSA better than glycated BSA and also showed weaker affinity (K_a = 23 μM) for glycated albumin compared with native state (K_a = 16 μM). Molecular docking study showed that both glyceraldehyde and colchicine bind to common residues located near Sudlow’s site I that explain the lower binding of colchicine in the glycated BSA. Based on our results, we believe that reduced drugs-binding affinity to glycated albumin may lead to drugs accumulation and precipitation in diabetic patients.     

The promiscuous protein binding ability of erythrosine B studied by metachromasy (metachromasia)

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly‐iodinated xanthene dye and an FDA‐approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein–protein interactions (PPIs) with a remarkably consistent median inhibitory concentration (IC₅₀) in the 5‐ to 30‐μM range. Because ErB exhibits metachromasy, that is, color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (K_d) and stoichiometry (n_b) using spectrophotometric methods. Binding was reversible, and the estimated affinities for both protein targets obtained here (K_d values of 14 and 20 μM for BSA and CD40L, respectively) were in good agreement with that expected from the PPI inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (n_b of 5–6 and 8–9 for BSA and CD40L, respectively), indicating the possibility of nonspecific binding of the flat and rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. Copyright © 2013 John Wiley & Sons, Ltd.     

Selective binding of cholesterol by recombinant fatty acid binding proteins

The sterol binding specificity of rat recombinant liver fatty acid binding protein (L-FABP) and intestinal fatty acid binding protein (I-FABP) was characterized with [3H]cholesterol and a fluorescent sterol analog dehydroergosterol. Ligand binding analysis, fluorescence spectroscopy, and activation of microsomal acyl-CoA:cholesterol acyltransferase activity showed that L-FABP-bound sterols. 1) Lipidex-1000 assay showed a dissociation constant Kd = 0.78 +/- 0.18 microM and stoichiometry of 0.47 +/- 0.16 mol/mol for [3H]cholesterol binding to L-PABP. 2) With [3H]cholesterol/phosphatidylcholine liposomes, the cholesterol binding parameters for L-FABP were Kd = 1.53 +/- 0.28 microM and stoichiometry 0.83 +/- 0.07 mol/mol. 3) L-FABP interaction with dehydroergosterol altered the fluorescence intensity and polarization of dehydroergosterol. Dehydroergosterol bound to L-FABP with Kd = 0.37 microM and a stoichiometry of 0.83 mol/mol. 4) Cholesterol and dehydroergosterol decreased L-FABP tyrosine lifetime. Dehydroergosterol binding produced sensitized emission of bound dehydroergosterol with longer lifetime.5) L-FABP bound two cis-parinaric acid molecules/molecule of protein. Cholesterol displaced one of these bound cis-parinaric acids. 6) L-FABP enhanced acyl-CoA:cholesterol acyltransferase in a concentration-dependent manner. In contrast, these assays indicated that I-FABP did not bind sterols. Thus, L-FABP appears able to bind 1 mol of cholesterol/mol of L-FABP, the L-FABP sterol binding site is equivalent to one of the two fatty acid binding sites, and L-FABP stimulates acyl-CoA:cholesterol acyltransferase by transfer of cholesterol.     

Two modes of fatty acid binding to bovine β-lactoglobulin--crystallographic and spectroscopic studies

Lactoglobulin is a natural protein present in bovine milk and common component of human diet, known for binding with high affinity wide range of hydrophobic compounds, among them fatty acids 12–20 carbon atoms long. Shorter fatty acids were reported as not binding to β‐lactoglobulin. We used X‐ray crystallography and fluorescence spectroscopy to show that lactoglobulin binds also 8‐ and 10‐carbon caprylic and capric acids, however with lower affinity. The determined apparent association constant for lactoglobulin complex with caprylic acid is 10.8 ± 1.7 × 10³ M^?1, while for capric acid is 6.0 ± 0.5 × 10³ M^?1. In crystal structures determined with resolution 1.9 Å the caprylic acid is bound in upper part of central calyx near polar residues located at CD loop, while the capric acid is buried deeper in the calyx bottom and does not interact with polar residues at CD loop. In both structures, water molecule hydrogen‐bonded to carboxyl group of fatty acid is observed. Different location of ligands in the binding site indicates that competition between polar and hydrophobic interactions is an important factor determining position of the ligand in β‐barrel. Copyright © 2010 John Wiley & Sons, Ltd.     

Combined spectroscopies and molecular docking approach to characterizing the binding interaction between lisinopril and bovine serum albumin

To further understand the mode of action and pharmacokinetics of lisinopril, the binding interaction of lisinopril with bovine serum albumin (BSA) under imitated physiological conditions (pH 7.4) was investigated using fluorescence emission spectroscopy, synchronous fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD) and molecular docking methods. The results showed that the fluorescence quenching of BSA near 338 nm resulted from the formation of a lisinopril–BSA complex. The number of binding sites (n) for lisinopril binding on subdomain IIIA (site II) of BSA and the binding constant were ~ 1 and 2.04 × 10⁴ M^–1, respectively, at 310 K. The binding of lisinopril to BSA induced a slight change in the conformation of BSA, which retained its α‐helical structure. However, the binding of lisinopril with BSA was spontaneous and the main interaction forces involved were van der Waal's force and hydrogen bonding interaction as shown by the negative values of ΔG⁰, ΔH⁰ and ΔS⁰ for the binding of lisinopril with BSA. It was concluded from the molecular docking results that the flexibility of lisinopril also played an important role in increasing the stability of the lisinopril–BSA complex. Copyright © 2015 John Wiley & Sons, Ltd.     

Insights into binding of fatty acids by fatty acid binding proteins

Members of the phylogenetically related intracellular lipid binding protein (iLBP) are characterized by a highly conserved tertiary structure, but reveal distinct binding preferences with regard to ligand structure and conformation, when binding is assessed by the Lipidex method (removal of unbound ligand by hydrophobic polymer) or by isothermal titration calorimetry, a true equilibrium method. Subfamily proteins bind retinoids, subfamily II proteins bind bulky ligands, examples are intestinal bile acid binding protein (I-BABP) and liver fatty acid binding protein (L-FABP) which binds 2 ligand molecules, preferably monounsaturated and n-3 fatty acids. Subfamily III intestinal fatty acid binding protein (I-FABP) binds fatty acid in a bent conformation. The fatty acid bound by subfamily IV FABPs has a U-shaped conformation; here heart (H-) FABP preferably binds n-6, brain (B-) FABP n-3 fatty acids. The ADIFAB-method is a fluorescent test for fatty acid in equilibrium with iLBP and reveals some correlation of binding affinity to fatty acid solubility in the aqueous phase; these data are often at variance with those obtained by the other methods. Thus, in this review published binding data are critically discussed, taking into account on the one hand binding increments calculated for fatty acid double bonds on the basis of the solubility hypothesis, on the other hand the interpretation of calorimetric data on the basis of crystallographic and solution structures of iLBPs.     

FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steady-state and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantum yield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant K_A = 7.0(± 0.8) × 10⁴ M^− 1. Taken together, the FAD–protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interaction was performed among alternative NADH dehydrogenases.     

Probing the binding of two 19‐nortestosterone derivatives to human serum albumin: insights into the interactions of steroid hormone drugs with functional biomacromolecule

Norethindrone acetate (NETA) is a fatty acid ester of norethindrone (NET) that can convert to its more active parent compound NET when orally administered. To study the interactions of NETA and NET with human serum albumin (HSA), we applied fluorescence spectroscopy, circular dichroism (CD), and molecular docking. The effects of metal ions on the HSA–NETA/NET system were also explored. Fluorescence data showed that the quenching mechanism of HSA by NETA and NET was consistent with a static model and that the binding constant of NETA was higher than that of NET. Thermodynamic parameters indicated that hydrogen bonds and van der Waals forces were the main forces maintaining the stability of the HSA–NETA/NET complex. Molecular modeling studies revealed that NETA and NET were bound within subdomain IIA of HSA, in accordance with the site probe results. Synchronous fluorescence spectroscopy, CD, and three‐dimensional fluorescence spectroscopy further confirmed that the binding of NETA/NET to HSA changed the secondary structure of the protein. All other metal ions, except for Ca²⁺, decreased the K value of the HSA–NETA/NET system with enhancement of the maximum effectiveness of NETA/NET. Three commercially available steroid hormone drugs influenced the binding ability of NETA on HSA to different extents. This study provides novel insights into the interactions between HSA and NETA/NET, as well as a solid foundation for future research on drug pharmacokinetics and pharmacodynamics. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization of the binding of neomycin/paromomycin sulfate with DNA using acridine orange as fluorescence probe and molecular docking technique

The binding of neomycin sulfate (NS)/paromomycin sulfate (PS) with DNA was investigated by fluorescence quenching using acridine orange (AO) as a fluorescence probe. Fluorescence lifetime, FT-IR, circular dichroism (CD), relative viscosity, ionic strength, DNA melting temperature, and molecular docking were performed to explore the binding mechanism. The binding constant of NS/PS and DNA was 6.70 × 10³/1.44 × 10³ L mol^?1 at 291 K. The values of ΔH^θ, ΔS^θ, and ΔG^θ suggested that van der Waals force or hydrogen bond might be the main binding force between NS/PS and DNA. The results of Stern–Volmer plots and fluorescence lifetime measurements all revealed that NS/PS quenching the fluorescence of DNA–AO was static in nature. FT-IR indicated that the interaction between DNA and NS/PS did occur. The relative viscosity and melting temperature of DNA were almost unchanged when NS/PS was introduced to the solution. The fluorescence intensity of NS/PS–DNA–AO was decreased with the increase in the ionic strength. For CD spectra of DNA, the intensity of positive band at nearly 275 nm was decreased and that of negative band at nearly 245 nm was increased with the increase in the concentration of NS/PS. The binding constant of NS/PS with double-stranded DNA (dsDNA) was larger than that of NS/PS with single-stranded DNA (ssDNA). From these studies, the binding mode of NS/PS with DNA was evaluated to be groove binding. The results of molecular docking further indicated that NS/PS could enter into the minor groove in the A–T rich region of DNA.     

Destructive effect of anticancer oxali-palladium on heme degradation through the generation of endogenous hydrogen peroxide

The interaction between human hemoglobin (Hb) and oxali-palladium was studied using different spectroscopic methods of UV–vis, fluorescence, circular dichroism (CD), and chemiluminescence at two temperatures of 25 and 37°C. The experimental results showed that both dynamic and static quenching is occurred simultaneously when oxali-palladium quenches the fluorescence of Hb. According to the fluorescence quenching method, the binding site number, apparent binding constant, and corresponding thermodynamic parameters were measured at two temperatures. The values of ΔH°, ΔS°, and ΔG° indicate that process of the formation of oxali-palladium–Hb complex is a spontaneous interaction procedure in which electrostatic interaction plays a major role. In addition, UV–vis and CD results showed that the addition of oxali-palladium changes the conformation of Hb. To evaluate the functional changes of Hb via destruction of the heme structure, fluorescence studies were performed. The results demonstrated that two fluorescent heme degradation products are found during the interaction of oxali-palladium with Hb. Also, the amount of hydrogen peroxide produced in the solution of Hb due to the interaction of oxali-palladium with Hb using chemiluminescence method indicated heme degradation in the protein is occurred. Structural and functional changes induced in Hb via heme degradation are considered as side effects of this synthesized anticancer drug.     

Binding of heparin to antithrombin III: The use of dansyl and rhodamine labels

Low molecular weight heparin of low-anticoagulant activity and high molecular weight heparin of correspondingly high activity were prepared by chromatography on protamine-Sepharose; preparations subjected to limited N-desulfation (5–10% free amino groups) by solvolysis were labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) or rhodamine B isothiocyanate (RITC). The fluorescent heparins retained approximately 50% of the original anticoagulant activities. Dansyl-heparin on binding to antithrombin III (ATIII) exhibited a 2.5-fold enhancement of dansyl fluorescence intensity. This effect could be prevented by excess unlabeled heparin. A 7900 molecular weight dansyl-heparin preparation bound to ATIII with a stoichiometry of close to 2:1 and with an apparent association constant for binding (K_a) of 4.9 × 10⁵, m^?1, whereas a 21,600 molecular weight fraction bound at 0.7:1 with the protein and with an apparent K_a = 7.9 × 10⁵, m^?1. When ATIII reacted with a mixture of low molecular weight dansyl-heparin and low molecular weight RITC-heparin, there was enhancement of RITC fluorescence emission when excited at the dansyl excitation maximum; this effect was not observed when either of the labeled heparin species was prepared from high molecular weight material. The results are consistent with the proposal that a single molecule of high molecular weight, high-activity heparin occupies two sites when it binds to ATIII, whereas low molecular weight, low-activity heparin binds to the two sites separately.     

Combined spectroscopic and molecular docking approach to probing binding interactions between lovastatin and calf thymus DNA

The binding interaction of lovastatin with calf thymus DNA (ct‐DNA) was studied using UV/Vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD), viscosity measurement and molecular docking methods. The experimental results showed that there was an obvious binding interaction of lovastatin with ct‐DNA and the binding constant (K_b) was 5.60 × 10³ M^–1 at 298 K. In the binding process of lovastatin with ct‐DNA, the enthalpy change (ΔH⁰) and entropy change (ΔS⁰) were –24.9 kJ/mol and –12.0 J/mol/K, respectively, indicating that the main binding interaction forces were van der Waal's force and hydrogen bonding. The molecular docking results suggested that lovastatin preferred to bind on the minor groove of different B‐DNA fragments and the conformation change of lovastatin in the lovastatin–DNA complex was obviously observed, implying that the flexibility of lovastatin molecule plays an important role in the formation of the stable lovastatin–ct‐DNA complex. Copyright © 2015 John Wiley & Sons, Ltd.     

Probing the structural interactions between methotrexate and dexamethasone with muscle cystatin: a biophysical study

Abstract

Drug protein interactions have gained considerable attention over the past many years. In the current communication the association of muscle cystatin (MC) with anti-rheumatic drugs methotrexate and dexamethasone was studied by thiol proteinase inhibitor assay, ultra violet (UV) absorption, fluorescence spectroscopy, and fluorescence transform infra-red spectroscopy (FTIR). A static pattern of quenching was noticed between muscle cystatin and methotrexate (MTX). Binding constant (K_a) of methotrexate to muscle cystatin was found to be 1?×?10^?7 M^?1 and the stoichiometry of binding was calculated to be one. Fluorescence measurement of the emission quenching reveals that the quenching process of cystatin by dexamethasone (DXN) was also static. The stoichiometry of binding and binding constant was also obtained. Additional evidence regarding MTX–MC and DXN–MC was obtained from UV spectroscopy and FTIR spectroscopic results. Such spectroscopic studies would help in modelling new candidate drugs for rheumatoid arthritis based on their cystatin binding profile.

Communicated by Ramaswamy H. Sarma     

Thermodynamics of ligand binding to histone deacetylase like amidohydrolase from Bordetella/Alcaligenes

Thermodynamic studies on ligand–protein binding have become increasingly important in the process of drug design. In combination with structural data and molecular dynamics simulations, thermodynamic studies provide relevant information about the mode of interaction between compounds and their target proteins and therefore build a sound basis for further drug optimization. Using the example of histone deacetylases (HDACs), particularly the histone deacetylase like amidohydrolase (HDAH) from Bordetella/Alcaligenes, a novel sensitive competitive fluorescence resonance energy transfer‐based binding assay was developed and the thermodynamics of interaction of both fluorescent ligands and inhibitors to histone deacetylase like amidohydrolase were investigated. The assay consumes only small amounts of valuable target proteins and is suitable for fast kinetic and mechanistic studies as well as high throughput screening applications. Binding affinity increased with increasing length of aliphatic spacers (n = 4–7) between the hydroxamate moiety and the dansyl head group of ligand probes. Van't Hoff plots revealed an optimum in enthalpy contribution to the free energy of binding for the dansyl‐ligand with hexyl spacer. The selectivity in the series of dansyl‐ligands against human class I HDAC1 but not class II HDACs 4 and 6 increased with the ratio of ΔH⁰/ΔG⁰. The data clearly emphasize the importance of thermodynamic signatures as useful general guidance for the optimization of ligands or rational drug design. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of ligand binding on the stability of aldo–keto reductases: Implications for stabilizer or destabilizer chaperones

Ligands such as enzyme inhibitors stabilize the native conformation of a protein upon binding to the native state, but some compounds destabilize the native conformation upon binding to the non‐native state. The former ligands are termed “stabilizer chaperones” and the latter ones “destabilizer chaperones.” Because the stabilization effects are essential for the medical chaperone (MC) hypothesis, here we have formulated a thermodynamic system consisting of a ligand and a protein in its native‐ and non‐native state. Using the differential scanning fluorimetry and the circular dichroism varying the urea concentration and temperature, we found that when the coenzyme NADP⁺ was absent, inhibitors such as isolithocholic acid stabilized the aldo–keto reductase AKR1A1 upon binding, which showed actually the three‐state folding, but destabilized AKR1B10. In contrast, in the presence of NADP⁺, they destabilized AKR1A1 and stabilized AKR1B10. To explain these phenomena, we decomposed the free energy of stabilization (ΔΔG) into its enthalpy (ΔΔH) and entropy (ΔΔS) components. Then we found that in a relatively unstable protein showing the three‐state folding, native conformation was stabilized by the negative ΔΔH in association with the negative ΔΔS, suggesting that the stabilizer chaperon decreases the conformational fluctuation of the target protein or increase its hydration. However, in other cases, ΔΔG was essentially determined by the delicate balance between ΔΔH and ΔΔS. The proposed thermodynamic formalism is applicable to the system including multiple ligands with allosteric interactions. These findings would promote the development of screening strategies for MCs to regulate the target conformations.     

Locating the binding sites of retinol and retinoic acid with milk β-lactoglobulin

β-lactoglobulin (β-LG) is a member of lipocalin superfamily of transporters for small hydrophobic molecules such as retinoids. We located the binding sites of retinol and retinoic acid on β-LG in aqueous solution at physiological conditions, using FTIR, CD, fluorescence spectroscopic methods, and molecular modeling. The retinoid-binding sites and the binding constants as well as the effect of retinol and retinoic acid complexation on protein stability and secondary structure were determined. Structural analysis showed that retinoids bind strongly to β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K _{retinol- β -LG?}=?6.4 (±?.6)?×?10⁶?M^?1 and K _{retinoic acid- β -LG?}=?3.3 (±?.5)?×?10⁶?M^?1. The number of retinoid molecules bound per protein (n) is 1.1 (±?.2) for retinol and 1.5 (±?.3) for retinoic acid. Molecular modeling showed the participation of several amino acids in the retinoid–protein complexes with the free binding energy of ?8.11?kcal/mol for retinol and ?7.62?kcal/mol for retinoic acid. Protein conformation was altered with reduction of β-sheet from 59 (free protein) to 52–51% and a major increase in turn structure from 13 (free protein) to 24–22%, in the retinoid–β-LG complexes, indicating a partial protein destabilization.     

Multi-spectroscopic and molecular modelling approach to investigate the interaction of riboflavin with human serum albumin

Riboflavin (RF) plays an important role in various metabolic redox reactions in the form of flavin adenine dinucleotide and flavin mononucleotide. Human serum albumin (HSA) is an important protein involved in the transportation of drugs, hormones, fatty acid and other molecules which determine the biodistribution and physiological fate of these molecules. In this study, we have investigated the interaction of riboflavin RF with HSA under simulative physiological conditions using various biophysical, calorimetric and molecular docking techniques. Results demonstrate the formation of riboflavin–HSA complex with binding constant in the order of 10⁴ M^?1. Fluorescence spectroscopy confirms intermediate strength having a static mode of quenching with stoichiometry of 1:1. Experimental results suggest that the binding site of riboflavin mainly resides in sub-domain IIA of HSA and that ligand interaction increases the α-helical content of HSA. These parameters were further verified by isothermal titration calorimetry ITC which confirms the thermodynamic parameters obtained by fluorescence spectroscopy. Molecular docking was employed to suggest a binding model. Based on thermodynamic, spectroscopic and computational observations it can be concluded that HSA-riboflavin complex is mainly stabilized by various non-covalent forces with binding energy of ?7.2 kcal mol^?1.     

X-ray crystallographic analysis of adipocyte fatty acid binding protein (aP2) modified with 4-hydroxy-2-nonenal

Fatty acid binding proteins (FABP) have been characterized as facilitating the intracellular solubilization and transport of long‐chain fatty acyl carboxylates via noncovalent interactions. More recent work has shown that the adipocyte FABP is also covalently modified in vivo on Cys117 with 4‐hydroxy‐2‐nonenal (4‐HNE), a bioactive aldehyde linked to oxidative stress and inflammation. To evaluate 4‐HNE binding and modification, the crystal structures of adipocyte FABP covalently and noncovalently bound to 4‐HNE have been solved to 1.9 Å and 2.3 Å resolution, respectively. While the 4‐HNE in the noncovalently modified protein is coordinated similarly to a carboxylate of a fatty acid, the covalent form show a novel coordination through a water molecule at the polar end of the lipid. Other defining features between the two structures with 4‐HNE and previously solved structures of the protein include a peptide flip between residues Ala36 and Lys37 and the rotation of the side chain of Phe57 into its closed conformation. Representing the first structure of an endogenous target protein covalently modified by 4‐HNE, these results define a new class of in vivo ligands for FABPs and extend their physiological substrates to include bioactive aldehydes.