Specific interaction of chalcone-protein: cardamonin binding site II on the human serum albumin molecule

Cardamonin (2',4'-dihydroxy-6'-methoxychalcone), one of the main constituents from the seeds of Alpinia katsumadai Hayata, belongs to chalcone with its antibacterial, antiinflammatory and other important therapeutic activities of significant potency and low systemic toxicity. In this article, the interaction of cardamonin to human serum albumin (HSA) has been studied for the first time by spectroscopic methods including Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD), and UV-absorption spectroscopy in combination with fluorescence quenching under physiological conditions with drug concentrations of 0.67-4.0 microM. The results of the spectroscopic measurements and the thermodynamic parameters obtained (the enthalpy change DeltaH(0) and the entropy change DeltaS(0) were calculated to be -25.312 and 7.040 J.mol(-1).K(-1) according to the van't Hoff equation) suggest that hydrophobic interaction is the predominant intermolecular forces stabilizing the complex, which is also in good agreement with the results of the molecule modeling study. The alterations of protein secondary structure in the presence of cardamonin in aqueous solution were quantitatively calculated by the evidence from CD and FTIR spectroscopes with reductions of alpha-helices of about 20%, decreases of beta-sheet structures of about 14%, and increases of beta-turn structures of about 15%. The quenching mechanism and the number of binding sites (n approximately 1) were obtained by fluorescence titration data. Fluorescent displacement measurements confirmed that cardamonin binds HSA on site II. In addition, the effects of common ions on the constants of the cardamonin-HSA complex were also discussed.     

The oxalate effect on release of iron from human serum transferrin explained

A unique feature of the mechanism of iron binding to the transferrin (TF) family is the synergistic relationship between metal binding and anion binding. Little or no iron will bind to the protein without concomitant binding of an anion, physiologically identified as carbonate. Substitution of oxalate for carbonate produces no significant changes in polypeptide folding or domain orientation in the N-lobe of human serum TF (hTF) as revealed by our 1.2A structure. The oxalate is able to bind to the iron in a symmetric bidentate fashion, which, combined with the low pK(a) of the oxalate anion, makes iron displacement more difficult as documented by both iron release kinetic and equilibrium data. Characterization of an N-lobe in which the arginine at position 124 is mutated to alanine reveals that the stabilizing effect of oxalate is even greater in this mutant and nearly cancels the destabilizing effect of the mutation. Importantly, incorporation of oxalate as the synergistic anion appears to completely inhibit removal of iron from recombinant full-length hTF by HeLa S(3) cells, strongly indicating that oxalate also replaces carbonate in the C-lobe to form a stable complex. Kinetic studies confirm this claim. The combination of structural and functional data provides a coherent delineation of the effect of oxalate binding on hTF and rationalizes the results of many previous studies. In the context of iron uptake by cells, substitution of carbonate by oxalate effectively locks the iron into each lobe of hTF, thereby interfering with normal iron metabolism.     

Secondary structure of globulins from plant seeds: a re-evaluation from circular dichroism measurements

The secondary structure parameters of plant seed globulins (11S from Brassica napus L, 11S from Helianthus annuus L, IIS from Vicia faba, 7S from Phaseolus vulgaris L) have been determined from their circular dichroism spectra by the method of Provencher and Glöckner. According to this method, the proteins contain 40–50% β-sheet structure and only about 10% helical structure. We conclude, therefore, that the plant seed globulins belong to the class of β-sheet proteins. Their overall secondary structure is homologous. It is shown that the method of Provencher and Glöckner provides reasonable secondary structure parameters for proteins which are rich in β-sheet structure even if the spectral range utilized for analysis is restricted to 210–240 nm.     

Deciphering the binding of carbendazim (fungicide) with human serum albumin: A multi-spectroscopic and molecular modelling studies

Carbendazim is a benzimidazole fungicide used to control the fungal invasion. However, its exposure might lead to potential health problems. The present study evaluates the interaction of carbendazim (CAR) with human serum albumin (HSA) which is an important drug carrier protein and plays a very crucial role in the transportation of small molecules. A number of biophysical techniques were employed to investigate the binding of CAR with HSA. The increased UV-absorption of HSA on titrating with CAR suggests the formation of HSA–CAR complex and it could be due to the exposure of aromatic residues. The fluorescence study confirmed that CAR quenches the fluorescence of HSA and showed the static mode of quenching. CAR (50 µM) quenches around 56.14% of the HSA fluorescence. The quenching constant, binding constant, number of binding site and free energy change was calculated by fluorescence quenching experiment. Competitive displacement assay showed Sudlow’s site I as the primary binding site of CAR on HSA. The synchronous fluorescence study revealed the perturbation in the microenvironment around tyrosine and tryptophan residues upon binding of CAR to HSA. The circular dichroism results suggested that the binding of CAR to HSA altered its secondary structure. Molecular docking experiment demonstrated the binding of CAR to Sudlow’s site I of HSA. Docking studies suggested that the hydrogen bonding, van der Waals and pi-alkyl are playing role in the interaction of CAR with HSA. The study confirmed the conformational changes within HSA upon binding of CAR.     

Effects of organic and inorganic polyamine cations on the structure of human serum albumin

The presence of several high affinity binding sites on human serum albumin (HSA) makes it a possible target for many organic and inorganic molecules. Organic polyamines are widely distributed in living cells and their biological roles have been associated with their physical and chemical interactions with proteins, nucleic acids, and lipids. This study is designed to examine the effects of spermine, spermidine, putrescine, and cobalt [Co(III)]-hexamine cations on the solution structure of HSA using Fourier transform IR, UV-visible, and circular dichroism (CD) spectroscopic methods. The spectroscopic results show that polyamine cations are located along the polypeptide chains with no specific interaction. The order of perturbations is associated with the number of positive charges of the polyamine cation: spermine > Co(III)-hexamine > spermidine > putrescine. The overall binding constants are 1.7 x 10(4), 1.1 x 10(4), 5.4 x 10(3), and 3.9 x 10(3)M(-1), respectively. The protein conformation is altered (IR and CD data) with reductions of alpha helices from 60 to 55% for free HSA to 50-40% and with increases of beta structures from 22 to 15% for free HSA to 33-23% in the presence of polyamine cations.     

Experimental and theoretical studies of the three-dimensional structure of human interleukin-4

The structure of human interleukin 4 (IL-4) was predicted utilizing a series of experimental and theoretical techniques. Circular Dichroism (CD) spectroscopy indicated that IL-4 belonged to the all alpha-helix class of protein structures. Secondary structure prediction, site-directed mutagenesis, and CD spectroscopy suggested a predominantly alpha-helical structure, consistent with a four-helix bundle structural motif. A human/mouse IL-4 chimera was constructed to qualitatively evaluate alternative secondary structure predictions. The four predicted helices were assembled into tertiary structures using established algorithms. The mapping of three disulfide bridges in IL-4 provided additional constraints on possible tertiary structures. Using accessible surface contact area as a criterion, the most suitable structures were right handed all antiparallel four-helix bundles with two overhand loop connections. Successful loop closure and incorporation of the three disulfide constraints were possible while maintaining the expected shape, solvent accessibility, and steric interactions between loops and helices. Lastly, energy minimization was used to regularize the chain.     

Spectral magnitude effects on the analyses of secondary structure from circular dichroism spectroscopic data

The effects of spectral magnitude on the calculated secondary structures derived from circular dichroism (CD) spectra were examined for a number of the most commonly used algorithms and reference databases. Proteins with different secondary structures, ranging from mostly helical to mostly beta-sheet, but which were not components of existing reference databases, were used as test systems. These proteins had known crystal structures, so it was possible to ascertain the effects of magnitude on both the accuracy of determining the secondary structure and the goodness-of-fit of the calculated structures to the experimental data. It was found that most algorithms are highly sensitive to spectral magnitude, and that the goodness-of-fit parameter may be a useful tool in assessing the correct scaling of the data. This means that parameters that affect magnitude, including calibration of the instrument, the spectral cell pathlength, and the protein concentration, must be accurately determined to obtain correct secondary structural analyses of proteins from CD data using empirical methods.     

Prediction of the tertiary structure of the alpha-subunit of tryptophan synthase

The tertiary structure of the alpha-subunit of tryptophan synthase was proposed using a combination of experimental data and computational methods. The vacuum-ultraviolet circular dichroism spectrum was used to assign the protein to the alpha/beta-class of supersecondary structures. The two-domain structure of the alpha-subunit (Miles et al.: Biochemistry 21:2586, 1982; Beasty and Matthews: Biochemistry 24:3547, 1985) eliminated consideration of a barrel structure and focused attention on a beta-sheet structure. An algorithm (Cohen et al.: Biochemistry 22:4894, 1983) was used to generate a secondary structure prediction that was consistent with the sequence data of the alpha-subunit from five species. Three potential secondary structures were then packed into tertiary structures using other algorithms. The assumption of nearest neighbors from second-site revertant data eliminated 97% of the possible tertiary structures; consideration of conserved hydrophobic packing regions on the beta-sheet eliminated all but one structure. The native structure is predicted to have a parallel beta-sheet flanked on both sides by alpha-helices, and is consistent with the available data on chemical cross-linking, chemical modification, and limited proteolysis. In addition, an active site region containing appropriate residues could be identified as well as an interface for beta 2-subunit association. The ability of experimental data to facilitate the prediction of protein structure is discussed.     

Protein binding investigation by difference circular dichroism: Native and acetylated human serum albumins

A modified albumin was prepared by selective reaction of Lys₁₉₉ with acetyl salicylic acid. Protein binding investigation was carried out on native and modified proteins by difference circular dichroism (ΔCD). Acetylation of Lys₁₉₉ reduces significantly the effects of the interaction between drugs in the stereoselective HSA binding at specific binding areas. © 1995 Wiley-Liss, Inc.     

Expression,purification and characterisation of a novel mutant of the human protein kinase CK2

The recombinant catalytic subunit of human protein kinase CK2 bas been mutagenised at the C-terminal region in an attempt to induce this tail to fold. We suppose in fact that this unstructured C-terminus just might be responsible for the high degradability of the human enzyme. On the basis of theoretical calculations we choose to substitute two distal prolines with alanines (PA 382-384). The mutant bas been purified to the electrophoretic homogeneity by means of three chromatographic steps. By circular dichroism Spectroscopy we verified if the double amino acids substitution reflected on the secondary structure of the recombinant subunit. According to our theoretical predictions, we observed that the -helix content of the protein increased when the two distal prolines were substituted by alanines. Moreover the mutant catalytic subunit shows a reduced ability to bind a classical inhibitor such as heparin.     

Effect of guanidine hydrochloride and urea on the interaction of 6-thioguanine with human serum albumin: a spectroscopic and molecular dynamics based study

6-thioguanine (6-TG) is an antineoplastic, nucleobase guanine, purine analog drug belongs to thiopurine drug-family of antimetabolites. In the present study, we report an experimental approach towards interaction mechanism of 6-TG with human serum albumin (HSA) and examine the chemical stability of HSA in the presence of denaturants such as guanidine hydrochloride (GdnHCl) and urea. Interaction of 6-TG with HSA has been studied by various spectroscopic and spectropolarimeteric methods to investigate what short of binding occurs at physiological conditions. 6-TG binds in the hydrophobic cavity of subdomain IIA of HSA by static quenching mechanism which induces conformation alteration in the protein structure. That helpful for further study of denaturation process where change in secondary structures causes unfolding of protein that also responsible for severance of domain III from rest of the protein part. We have also performed molecular simulation and molecular docking study in the presence of denaturating agents to determine the binding property of 6-TG and the effect of denaturating agents on the structural activity of HSA. We had found that GdnHCl is more effective denaturating agent when compared to urea. Hence, this study provides straight evidence of the binding mechanism of 6-TG with HSA and the formation of intermediate or unfolding transition that causes unfolding of HSA.     

Changes in secondary structure follow the dissociation of human insulin hexamers: a circular dichroism study

Vacuum UV circular dichroism spectra measured down to 178 nm for hexameric 2-zinc human insulin, zinc-free human insulin, and the two engineered and biologically active monomeric mutants, [B/S9D] and [B/S9D,T27E] human insulin, show significant differences. The secondary structure analysis of the 2-zinc human insulin (T6) in neutral solution was determined: 57% helix, 1% beta-strand, 18% turn, and 24% random coil. This is very close to the corresponding crystal structure showing that the solution and solid structures are similar. The secondary structure of the monomer shows a 10-15% increase in antiparallel beta-structure and a corresponding reduction in random coil structure. These structural changes are consistent with an independent analysis of the corresponding difference spectra. The advantage of secondary structure analyses of difference spectra is that the contribution of odd spectral features stemming mainly from side chain chromophores is minimized and the sensitivity of the analyses improved. Analysis of the CD spectra of T6 2-zinc, zinc-free human insulin and monomeric mutant insulin by singular value decomposition indicates that the secondary structure changes following the dissociation of hexamers into dimers and monomers are two-state processes.     

Influence of quercetin on the interaction of gliclazide with human serum albumin – spectroscopic and docking approaches

Protein‐binding interactions are displacement reactions which have been implicated as the causative mechanisms in many drug–drug interactions. Thus, the aim of presented study was to analyse human serum albumin‐binding displacement interaction between two ligands, hypoglycaemic drug gliclazide and widely distributed plant flavonoid quercetin. Fluorescence analysis was used in order to investigate the effect of substances on intrinsic fluorescence of human serum albumin (HSA) and to define binding and quenching properties of ligand–albumin complexes in binary and ternary systems, respectively. Both ligands showed the ability to bind to HSA, although to a different extent. The displacement effect of one ligand from HSA by the other one has been described on the basis of the quenching curves and binding constants comparison for the binary and ternary systems. According to the fluorescence data analysis, gliclazide presents a substance with a lower binding capacity towards HSA compared with quercetin. Results also showed that the presence of quercetin hindered the interaction between HSA and gliclazide, as the binding constant for gliclazide in the ternary system was remarkably lower compared with the binary system. This finding indicates a possibility for an increase in the non‐bound fraction of gliclazide which can lead to its more significant hypoglycaemic effect. Additionally, secondary and tertiary structure conformational alterations of HSA upon binding of both ligands were investigated using synchronous fluorescence, circular dichroism and FT‐IR. Experimental data were complemented with molecular docking studies. Obtained results provide beneficial information about possible interference upon simultaneous co‐administration of the food/dietary supplement and drug.     

Chiral recognition of metalaxyl enantiomers by human serum albumin: evidence from molecular modeling and photophysical approach

Metalaxyl is an acylamine fungicide, belonging to the most widely known member of the amide group. This task is aimed to scrutinize binding region and spatial structural change of principal vector human serum albumin (HSA) complex with (R)-/(S)-metalaxyl by exploiting molecular modeling, steady-state and time-resolved fluorescence, and circular dichroism (CD) approaches. According to molecular modeling, (R)-metalaxyl is situated within subdomains IIA and IIIA and the affinity of site I with (R)-metalaxyl is greater than site II, whereas (S)-metalaxyl is only located at subdomain IIA and the affinity of (S)-metalaxyl with site I is superior compared with that with (R)-metalaxyl. This coincides with the competitive ligand binding, guanidine hydrochloride-induced unfolding of protein, and hydrophobic 8-anilino-1-naphthalenesulfonic acid experiments; the acting forces between (R)-/(S)-metalaxyl and HSA are hydrophobic, π-π interactions, and hydrogen bonds, as derived from molecular modeling. Fluorescence emission manifested that the complex of (R)-/(S)-metalaxyl to HSA is the formation of adduct with an affinity of 10(4) M(-1), which corroborates the time-resolved fluorescence that the static type was operated. Furthermore, the changes of far-UV CD spectra evidence the polypeptide chain of HSA partially unfolded after conjugation with (R)-/(S)-metalaxyl. Through this work, we envisage that it can offer central clues on the biodistribution, absorption, and bioaccumulation of (R)-/(S)-metalaxyl.     

Study of the interaction between ofloxacin and human serum albumin by spectroscopic methods

The binding of ofloxacin (OFLX) to human serum albumin (HSA) was investigated by fluorescence and circular dichroism (CD) techniques. The binding parameters have been evaluated by a fluorescence quenching method. Competitive binding measurements were performed in the presence of warfarin and ibuprofen and suggest binding to the warfarin site I of HSA. The distance r between donor (HSA) and acceptor (OFLX) was estimated according to the Forster's theory of non‐radiatiative energy transfer. CD spectra revealed that the binding of OFLX to HSA induced conformational changes in HSA. Molecular docking was performed and shows that for the lowest energy complex OFLX is located in site I of HSA, which correlate to the competitive binding experiments. Copyright © 2011 John Wiley & Sons, Ltd.     

Probing the interaction of anticancer drug temsirolimus with human serum albumin: molecular docking and spectroscopic insight

The binding interaction between temsirolimus, an important antirenal cancer drug, and HSA, an important carrier protein was scrutinized making use of UV and fluorescence spectroscopy. Hyper chromaticity observed in UV spectroscopy in the presence of temsirolimus as compared to free HSA suggests the formation of complex between HSA and temsirolimus. Fluorescence quenching experiments clearly showed quenching in the fluorescence of HSA in the presence of temsirolimus confirming the complex formation and also confirmed that static mode of interaction is operative for this binding process. Binding constant values obtained through UV and fluorescence spectroscopy reveal strong interaction; temsirolimus binds to HSA at 298 K with a binding constant of 2.9 × 10⁴ M^?1implying the strength of interaction. The negative Gibbs free energy obtained through Isothermal titration calorimetry as well as quenching experiments suggests that binding process is spontaneous. Molecular docking further provides an insight of various residues that are involved in this binding process; showing the binding energy to be -12.9 kcal/mol. CD spectroscopy was retorted to analyze changes in secondary structure of HSA; increased intensity in presence of temsirolimus showing changes in secondary structure of HSA induced by temsirolimus. This study is of importance as it provides an insight into the binding mechanism of an important antirenal cancer drug with an important carrier protein. Once temsirolimus binds to HSA, it changes conformation of HSA which in turn can alter the functionality of this important carrier protein and this altered functionality of HSA can be highlighted in variety of diseases.     

Enantioselective inhibition of the binding of rac-profens to human serum albumin induced by lithocholate

The reversible binding of lithocholate to human serum albumin determines a decrease of the binding of rac-ketoprofen. The process was followed by displacement chromatography using increasing concentrations of the competitor, i.e., lithocholate, in the mobile phase. The inhibition of rac-ketoprofen binding resulting was enantioselective and greater displacement was observed for the (S) enantiomer. The displacement process resulting was competitive in nature, the two enantiomers of ketoprofen binding to the same binding site as the modifier. The investigation was extended to other nonsteroidal antiinflammatory drugs. The enantioselective binding inhibition was larger in the case of rac-naproxen and rac-suprofen with respect to the phenomenon observed in the case of rac-ketoprofen. The difference in circular dichroism spectroscopy was also used to characterize the binding of lithocholate to human serum albumin. This bile acid was proven to bind to site II on human serum albumin. The results, as obtained by displacement chromatography and difference circular dichroism spectroscopy, strongly support the hypothesized role of bile acids in inducing the enantioselective inhibition of ketoprofen binding to human serum albumin in patients suffering from liver diseases.     

G219S mutagenesis as a means of stabilizing conformational flexibility in the bacterial sodium channel NaChBac

The NaChBac sodium channel from Bacillus halodurans is a homologue of eukaryotic voltage-gated sodium channels. It can be solubilized in a range of detergents and consists of four identical subunits assembled as a tetramer. Sodium channels are relatively flexible molecules, adopting different conformations in their closed, open and inactivated states. This study aimed to design and construct a mutant version of the NaChBac protein that would insert into membranes and retain its folded conformation, but which would have enhanced stability when subjected to thermal stress. Modelling studies suggested a G219S mutant would have decreased conformational flexibility due to the removal of the glycine hinge around the proposed gating region, thereby imparting increased resistance to unfolding. The mutant expressed in Escherichia coli and purified in the detergent dodecyl maltoside was compared to wildtype NaChBac prepared in a similar manner. The mutant was incorporated into the membrane fraction and had a nearly identical secondary structure to the wildtype protein. When the thermal unfolding of the G219S mutant was examined by circular dichroism spectroscopy, it was shown to not only have a Tm ～10°C higher than the wildtype, but also in its unfolded state it retained more ordered helical structure than did the wildtype protein. Hence the G219S mutant was shown to be, as designed, more thermally stable.     

Investigation of the interaction between quercetin and human serum albumin by multiple spectra,electrochemical impedance spectra and molecular modeling

Quercetin (Qu), a flavonoid compound, exists widely in the human diet and exhibits a variety of pharmacological activities. This work is aimed at studying the effect of Qu on the bioactive protein, human serum albumin (HSA) under simulated biophysical conditions. Multiple spectroscopic methods (including fluorescence and circular dichroism), electrochemical impedance spectra (EIS) and molecular modeling were employed to investigate the interaction between Qu and HSA. The fluorescence quenching and EIS experimental results showed that the fluorescence quenching of HSA was caused by formation of a Qu–HSA complex in the ground state, which belonged to the static quenching mechanism. Based on the calculated thermodynamic parameters, it concluded that the interaction was a spontaneous process and hydrogen bonds combined with van der Waal's forces played a major role in stabilizing the Qu–HSA complex. Molecular modeling results demonstrated that several amino acids participated in the binding process and the formed Qu–HSA complex was stabilized by H‐bonding network at site I in sub‐domain IIA, which was further confirmed by the site marker competitive experiments. The evidence from circular dichroism (CD) indicated that the secondary structure and microenvironment of HSA were changed. Alterations in the conformation of HSA were observed with a reduction in the amount of α helix from 59.9% (free HSA) to 56% (Qu–HSA complex), indicating a slight unfolding of the protein polypeptides. Copyright © 2014 John Wiley & Sons, Ltd.     

Insights into the binding of paclitaxel to human serum albumin: multispectroscopic studies

The interaction of paclitaxel with human serum albumin (HSA) was studied using fluorescence, resonance light scattering, ultraviolet‐visible, circular dichroism and Fourier transform infrared spectroscopy at pH 7.4. Fluorescence data revealed that the fluorescence quenching of HSA by paclitaxel was a static quenching procedure. Time‐resolved fluorescence data also confirmed the quenching mode, which present a constant decay time of about 5 ns. The binding sites were approximately 1 and the binding constant suggested a weak association (324/M at 298 K), which is helpful for the release of the drug to targeted organs. The thermodynamic parameters, ΔG^○, ΔH° and ΔS° were calculated as – 1.06 × 10⁴ J/mol, 361 J/mol per K and 9.7 × 10⁴ J/mol respectively at 298 K, suggesting that binding was spontaneous and was driven mainly by hydrophobic interactions. The binding distance between HSA and paclitaxel was determined to be 2.23 nm based on the Förster theory. Analysis of circular dichroism, ultraviolet‐visible, three‐dimensional fluorescence, Fourier transform infrared and resonance light scattering spectra demonstrated that HSA conformation was slightly altered in the presence of paclitaxel and dimension of the individual HSA molecules were larger after interacting with paclitaxel. These results were confirmed by a molecular docking study. Copyright © 2013 John Wiley & Sons, Ltd.