Microenvironment Of Tryptophan Residues Research Articles

Exploring the binding mechanisms of thermally and ultrasonically induced molten globule-like β-lactoglobulin with heptanal as revealed by multi-spectroscopic techniques and molecular simulation

This work employed the model protein β-lactoglobulin (BLG) to investigate the contribution of microstructural changes to regulating the interaction patterns between protein and flavor compounds through employing computer simulation and multi-spectroscopic techniques. The formation of molten globule (MG) state-like protein during the conformational evolution of BLG, in response to ultrasonic (UC) and heat (HT) treatments, was revealed through multi-spectroscopic characterization. Differential MG structures were distinguished by variations in surface hydrophobicity and the microenvironment of tryptophan residues. Fluorescence quenching measurements indicated that the formation of MG enhanced the binding affinity of heptanal to protein. LC-MS/MS and NMR revealed the covalent bonding between heptanal and BLG formed by Michael addition and Schiff-base reactions, and MG-like BLG exhibited fewer chemical shift residues. Molecular docking and molecular dynamics simulation confirmed the synergistic involvement of hydrophobic interactions and hydrogen bonds in shaping BLG-heptanal complexes thus promoting the stability of BLG structures. These findings indicated that the production of BLG-heptanal complexes was driven synergistically by non-covalent and covalent bonds, and their interaction processes were influenced by processes-induced formation of MG potentially tuning the release and retention behaviors of flavor compounds.

Biomolecular interaction of purified recombinant Arabidopsis thaliana's alternative oxidase 1A with TCA cycle metabolites: Biophysical and molecular docking studies

In higher plants, the mitochondrial alternative oxidase (AOX) pathway plays an essential role in maintaining the TCA cycle/cellular carbon and energy balance under various physiological and stress conditions. Though the activation of AOX pathway upon exogenous addition of α-ketoacids/TCA cycle metabolites [pyruvate, α-ketoglutarate (α-KG), oxaloacetic acid (OAA), succinate and malic acid] to isolated mitochondria is known, the molecular mechanism of interaction of these metabolites with AOX protein is limited. The present study is designed to understand the biomolecular interaction of pure recombinant Arabidopsis thaliana AOX1A with TCA cycle metabolites under in vitro conditions using various biophysical and molecular docking studies. The binding of α-KG, fumaric acid and OAA to rAtAOX1A caused conformational change in the microenvironment of tryptophan residues as evidenced by red shift in the synchronous fluorescence spectra (∆λ = 60 nm). Besides, a decrease in conventional fluorescence emission spectra, tyrosine specific synchronous fluorescence spectra (∆λ = 15 nm) and α-helical content of CD spectra revealed the conformation changes in rAtAOX1A structure associated with binding of various TCA cycle metabolites. Further, surface plasmon resonance (SPR) and microscale thermophoresis (MST) studies revealed the binding affinity, while docking studies identified binding pocket residues, respectively, for these metabolites on rAtAOX1A.

Limited hydrolysis as a strategy to improve the non-covalent interaction of epigallocatechin-3-gallate (EGCG) with whey protein isolate near the isoelectric point

This study was aimed to investigate the interaction mechanisms and structural changes of whey protein isolate (WPI) and whey protein isolate hydrolysates (WPIHs) with epigallocatechin-3-gallate (EGCG) near the isoelectric point through multiple spectroscopic techniques and field emission scanning electron microscopy. Fluorescence spectra results indicated that limited hydrolysis endowed WPIHs with higher affinity for the EGCG but the increased degree of hydrolysis led to an opposite result. Thermodynamic analysis revealed that EGCG bound WPI primarily through hydrogen bonds and van der waals forces, while the hydrophobic force was the main driving force in the interaction of EGCG with WPIHs. Synchronous fluorescence and three-dimensional spectra confirmed that EGCG induced conformational alterations of WPI and WPIHs, which was further supported by Ultraviolet–Visible spectra. Raman spectra indicated that binding to EGCG resulted in changes in the microenvironment of tryptophan residues, CH bending vibration and the secondary structure arrangements of WPI and WPIHs. Furthermore, compared with a sheet-like structure of WPI-EGCG complexes, the morphology of WPIHs with limited hydrolysis presented an uneven blocky structure after complexing with EGCG. Our findings might be helpful to better understand the interactions of milk protein hydrolysates-EGCG and suggest the potential application of the formed complexes as bioactive ingredients in food industry.

Spectroscopic studies of binding interactions of 2-chloroethylphenyl sulphide with bovine serum albumin

Sulphur mustard is a lethal chemical warfare agent, known for its vesicant or blistering action on skin and mucous membrane. 2-chloroethylphenyl sulphide (CEPS) is a simulant of sulphur mustard. In the present study, the binding interaction of CEPS with bovine serum albumin (BSA) was investigated to determine its binding potential with skin proteins. The binding interactions studies were performed using UV–Vis absorbance, steady-state fluorescence, synchronous fluorescence, circular dichroism techniques. It is observed in UV–Vis absorption study, absorbance of BSA at 280 nm increases with a slight blue shift in the presence of CEPS indicated a complex formation between CEPS and BSA. Fluorescence quenching of emission intensity of BSA in the presence of CEPS indicated change in the microenvironment of fluorophores (Tryptophan residues). Stern-Volmer plot showed an inverse relationship between quenching (Ksv) and temperature. Thus, the quenching is operative via static quenching. The binding parameters suggested the role of non-covalent interactions and involvement of single set of equivalent binding sites. Change in thermodynamic parameters suggested that the binding is essentially enthalpy driven and spontaneous in nature. A synchronous fluorescence of BSA spectra in the presence of CEPS causes maximum change around the microenvironment of Tryptophan residues than Tyrosine residues. The intermolecular distance between the donor (BSA) and acceptor (CEPS) molecule was 3.5 nm as calculated from FRET analysis and indicated no significant transfer of energy between donor and acceptor. CD spectrum confirmed the changes in secondary structure of BSA upon binding with CEPS. The esterase enzyme activity of BSA in presence of CEPS showed a decrease in enzyme activity. The binding interactions were modeled in binding pockets using molecular docking calculations.

Phytochemical profile, antioxidant, α-amylase inhibition, binding interaction and docking studies of Justicia carnea bioactive compounds with α-amylase

The present study investigated the antioxidant and invitro antidiabetic capacities of Justicia carnea aqueous leaf extract (JCAE) using α-amylase inhibition model. α-Amylase binding-interaction with JCAE was also investigated using fluorescence spectroscopy and molecular docking. Phytochemical screening and Gas Chromatography-Mass Spectrometry (GC–MS) analysis indicated presence of bioactive compounds. Phenolic (132 mg GAE/g) and flavonoid contents (31.08 mg CE/g) were high. JCAE exhibited high antioxidant capacity and effectively inhibited α-amylase activity (IC50, 671.43 ± 1.88 μg/mL), though lesser than acarbose effect (IC50, 108.91 ± 0.61 μg/mL). α-Amylase intrinsic fluorescence was quenched in the presence of JCAE. Ultraviolet-visible and FT-IR spectroscopies affirmed mild changes in α-amylase conformation. Synchronous fluorescence analysis indicated alterations in the microenvironments of tryptophan residues near α-amylase active site. Molecular docking affirmed non-polar interactions of compounds 6 and 7 in JCAE with Asp-197 and Trp-58 residues of α-amylase, respectively. Overall, JCAE indicated potential to prevent postprandial hyperglycemia by slowing down carbohydrate hydrolysis.

Analysis of the structure-function relationship of alpha amylase complexed with polyacrylic acid

Alpha-amylase is frequently used in technologies that require its immobilization, stabilization or encapsulation. Polyacrylic acid is a very suitable polymer for these purposes because it can bind to enzymes and then be released under certain conditions without altering the functional capacity of enzymes. The consequences produced by polyacrylic acid on alpha-amylase structure and function have been investigated through various techniques. Calorimetric measurements allowed examining the nature of the binding reaction, stoichiometry and affinity, while spectroscopic techniques provided additional information about functional and structural perturbations of the enzyme. Isothermal titration calorimetry (ITC) revealed a mixed interaction and a binding model with a large number of molecules of protein per molecule of polyacrylic acid. One the one hand circular dichroism (CD) spectroscopy showed that alpha-amylase loses its secondary structure in the presence of increasing concentrations of polyacrylic acid, while it is stabilized by the polyelectrolyte at low pH. On the other hand, fluorescence spectra revealed that the three-dimensional enzyme structure was not affected in the microenvironment of tryptophan residues. Differential scanning calorimetry (DSC) thermograms showed that only one domain of alpha-amylase is affected in its conformational stability by the polymer. The unfolding process proved to be partially reversible. Finally, the enzyme retained more than 90 % of its catalytic activity even in excess of the polymer.

Quercetin-induced inactivation and conformational alterations of alpha-2-macroglobulin: multi-spectroscopic and calorimetric study

Quercetin is a widely used bioflavonoid found in onions, grapes, berries and citrus fruits. Under certain conditions, quercetin acts as a pro-oxidant thereby generating reactive oxygen species and promoting the oxidation of molecules. Our study investigates the effect of quercetin on the structure and function of alpha-2-macroglobulin (α2M) by employing various biophysical techniques and trypsin inhibitory assay. α2M is the major antiproteinase present in the plasma of vertebrates. Results of activity assay indicated that α2M loses its 56% of inhibitory activity on treatment with quercetin in the presence of light. UV spectroscopy reveals hyper chromaticity in absorption spectra of protein on interaction with quercetin suggesting structural change. The intrinsic fluorescence studies showed quenching of α2M spectra in the presence of quercetin, and the mode of quenching was found to be static in nature. Synchronous fluorescence indicated the alteration in the microenvironment of tryptophan residues. CD and FTIR spectroscopy confirms concentration-dependent alterations in secondary structure of α2M instigated by quercetin. The magnitude of binding constant, enthalpy change, entropy change and free energy change during the interaction process was determined by isothermal titration calorimetry. Hydrogen bonding and hydrophobic interaction were the main intermolecular forces involved during the process. This study identifies and signifies the damage induced by quercetin to α2M due to its pro-oxidant action. Communicated by Ramaswamy H. Sarma

Microenvironment of tryptophan residues in proteins of four structural classes: applications for fluorescence and circular dichroism spectroscopy

In this study we examined microenvironment of Trp residues in "dry" sets of nonhomologous proteins that belong to four structural classes, as well as in a "wet" set. In silico experiments showed that residues of Trp demonstrate higher surface accessibility in proteins of "alpha/beta" class where they are rarely included in beta strands. However, this feature has not caused "red" shift in fluorescence spectra in "alpha/beta" proteins in vitro, since there are several factors that should be combined together to cause it: high surface accessibility and high hydrophilicity of the microenvironment, the presence of destabilizing contacts with Asp, Asn, Leu, and multiple Tyr residues, as well as the lack of stabilizing interactions with Arg, Thr, and Pro. The occurrence of Trp residues has the highest value in beta-structural proteins, while they are not involved in aromatic-aromatic interactions with each other as frequently, as they do in proteins of "alpha + beta" class in which Trp residues are overrepresented near each other in the primary sequence. That is why the deformation of circular dichroism spectra because of Trp-Trp interactions is expected to be more frequent in proteins of "alpha + beta" class. In all four classes of proteins Trp residues are involved in long-range interactions with some hydrophobic (Leu, Val, Ile) and aromatic residues (Trp, Phe, and Tyr) more frequently than it is expected. They are involved in long-range interactions with some hydrophilic residues (Asp, Glu, Ser, and Lys) rarely than it is expected. Short-range interactions between Arg and Trp are overrepresented just in alpha-helical proteins.

Investigation of binding between fluoroquinolones and pepsin by fluorescence spectroscopy and molecular simulation.

In this paper, the interactions of pepsin with fluoroquinolones, including norfloxacin (NFX) or ofloxacin (OFX), were investigated using fluorescence spectroscopy. The effects of NFX or OFX on pepsin showed that the molecular conformation of pepsin and the microenvironment of tryptophan residues were changed under mimicked physiological conditions. Static quenching was suggested as a factor. Quenching constants and binding constants were determined and thermodynamic parameters were calculated at three temperatures (25°C, 31°C and 37°C). Molecular interaction distances (binding distance r) were obtained. Binding was enthalpy driven and the process was spontaneous. Synchronous fluorescence, three-dimensional fluorescence spectroscopy and molecular simulation were used for analysis. Interactions were further tested using molecular modelling. Quenching and binding constants of NFX with pepsin were the highest when testing NFX/OFX/fleroxacin/gatifloxacin with pepsin combinations. NFX was the strongest quencher, and affinity of NFX for pepsin was higher than that of OFX/fleroxacin/gatifloxacin.

A fluorometric study of modification of bovine serum albumin with structural analogues of taurine chloramine

We studied the effects of taurine chloramine and its structural analogues, antiplatelet agents, on the tertiary structure of bovine serum albumin by recording changes in its fluorescence spectrum. BSA was chosen as a model of the extracellular part of the purine P2Y12 receptor in platelets. For the detection of weak spectral shifts of the protein fluorescence, the index, which represents the ratio of the two light sums measured from approximately the middle of the spectrum towards the short- and longwave limits of its registration, was used. Administration of N-chlorotaurine and its analogues N-acetyl-N-chlorotaurine and N-isopropyl-N-chlorotaurine in albumin solution in equimolar concentrations cause weakening and red shift of the tryptophanyl fluorescence of the protein, indicating reorganization of the protein’s tertiary structure, thereby changing the properties of the microenvironment of tryptophan residues, primarily, Trp 134. All these changes are probably due to a covalent modification of the sulfhydryl group of Cys34 residue. It was hypothesized that chloramines of taurine have the ability to inhibit the activity of the ADP P2Y12 receptor in platelets due to the modification of its sulfhydryl group and that the inhibitory effect on the ADP-binding site has an allosteric character.

Binding of Janus kinase inhibitor tofacitinib with human serum albumin: multi-technique approach

In this report, we have investigated the binding affinity of tofacitinib with human serum albumin (HSA) under simulated physiological conditions by using UV–visible spectroscopy, fluorescence quenching measurements, dynamic light scattering (DLS), differential scanning calorimetry (DSC) and molecular docking methods. The obtained results demonstrate that fluorescence intensity of HSA gets quenched by tofacitinib and quenching occurs in static manner. Binding parameters calculated from modified Stern–Volmer equation shows that the drug binds to HSA with a binding constant in the order of 105. Synchronous fluorescence data deciphered the change in the microenvironment of tryptophan residue in HSA. UV spectroscopy and DLS measurements deciphered complex formation and reduction in hydrodynamic radii of the protein, respectively. Further DSC results show that tofacitinib increases the thermo stability of HSA. Hydrogen bonding and hydrophobic interaction are the main binding forces between HSA and tofacitinib as revealed by docking results.

Fluorescence investigations on choline phospholipid binding and chemical unfolding of HSP-1/2, a major protein of horse seminal plasma

Seminal fibronectin type-II (Fn-II) proteins interact with choline phospholipids present on the sperm plasma membrane and play a crucial role in sperm capacitation. Crystal structure of phosphorylcholine (PrC) complex of PDC-109, the major bovine Fn-II protein, together with fluorescence spectroscopic studies has shown that tryptophan residues are crucial for its specific interaction with choline phospholipids. In the present study, the heterogeneity and microenvironment of tryptophan residues in HSP-1/2, a major protein of horse seminal plasma (which is homologous to PDC-109) were investigated in the native state, in the presence of PrC and phosphatidylcholines (PCs) with short (valeryl, C-5) and long (myristoyl, C-14) chains, and upon denaturation using fluorescence quenching, time-resolved fluorescence and red-edge excitation shift (REES) measurements. The results obtained show that the environment of tryptophan residues in HSP-1/2 is more heterogeneous as compared to that in PDC-109. Binding of choline containing ligands afforded a protection to the tryptophan residues with the shielding order being: PrC≤divalaroyl PC<dimyristoyl PC. REES value obtained for HSP-1/2 (3.5nm) is smaller than that of observed for PDC-109 (4nm) and binding to PrC and DVPC reduced the REES to 1nm. HSP-1/2 exhibits only partial unfolding with chemical denaturants with no cooperativity, whereas complete unfolding was observed in the presence of 10mM dithiothreitol, indicating that disulfide linkages prevent complete unfolding of the protein. In the presence of PrC the transition midpoints shifted to higher concentrations of the denaturant together with a broadening of the sigmoidal transitions, indicating that ligand binding as well as polydispersity modulate the unfolding process.

Denaturation of proteins with beta-barrel topology induced by guanidine hydrochloride

The stability of the representatives of two protein classes with β-barrel topology: porcine odorant-binding protein (OBP) and a number of fluorescent proteins (FPs), was studied. It was shown that both of them are significantly more stable than globular α-helical and α/β proteins. At the same time the value of energy barrier between native and unfolded state for FPs exceeds that for OBP. It was found that the small guanidine hydrochloride concentrations induce local structural distur- bances in proteins: changes in microenvironment of tryptophan residue in the case of odorant-binding protein and decrease in chromophore non-planarity in the case of green fluorescent protein.

Interaction Behavior Between Niclosamide and Pepsin Determined by Spectroscopic and Docking Methods.

The interaction between niclosamide (NIC) and pepsin was investigated using multispectroscopic and molecular docking methods. Binding constant, number of binding sites, and thermodynamic parameters at different temperatures were measured. Results of fluorescence quenching and synchronous fluorescence spectroscopy in combination with three-dimensional fluorescence spectroscopy showed that changes occurred in the microenvironment of tryptophan residues and the molecular conformation of pepsin. Molecular interaction distance and energy-transfer efficiency between pepsin and NIC were determined based on Förster nonradiative energy-transfer mechanism. Furthermore, the binding of NIC inhibited pepsin activity in vitro. All these results indicated that NIC bound to pepsin mainly through hydrophobic interactions and hydrogen bonds at a single binding site. In conclusion, this study provided substantial molecular-level evidence that NIC could induce changes in pepsin structure and conformation.

Tryptophan residue of the D-galactose/D-glucose-binding protein from E. Coli localized in its active center does not contribute to the change in intrinsic fluorescence upon glucose binding.

Changes of the characteristics of intrinsic tryptophan fluorescence of the wild type of D-galactose/D-glucose-binding protein from Escherichia coli (GGBPwt) induced by D-glucose binding were examined by the intrinsic UV-fluorescence of proteins, circular dyhroism in the near-UV region, and acrylamide-induced fluorescence quenching. The analysis of the different characteristics of GGBPwt and its mutant form GGBP-W183A together with the analysis of the microenvironment of tryptophan residues of GGBPwt revealed that Trp 183, which is directly involved in sugar binding, has the least influence on the provoked by D-glucose blue shift and increase in the intensity of protein intrinsic fluorescence in comparison with other tryptophan residues of GGBP.

Thermodynamic study of the effects of ethanol on the interaction of ochratoxin A with human serum albumin

Ethanol effect on the interaction of ochratoxin A (OTA) with human serum albumin (HSA) was investigated by using fluorescence spectroscopy and Raman spectroscopy. The Raman results showed that after the binding of OTA, the microenvironment of tryptophan residue on HSA became less hydrophobic. The fluorescence quenching observations revealed that the binding constant for the binding of OTA to HSA decreased as ethanol concentration increased. The thermodynamic studies showed that the binding process of OTA to HSA switched from being entropy-driven to enthalpy-driven in the presence of increasing concentrations (0.7–24.7%, vol/vol) of ethanol. Enthalpy–entropy compensation effect for the binding of OTA to HSA in the presence of different ethanol concentrations had been found. Based on the thermodynamic analyses, we concluded that the ethanol-induced variation of the shape of binding site of OTA on HSA and the solvent reorganization surrounding the OTA–HSA complex are the two dominant effects.

DENATURATION OF HUMAN SERUM ALBUMIN BY CERIUM (III) CHLORIDE

Cerium (III) Chloride-induced conformational changes of human serum albumin, HSA, in phosphate buffer, 10 mM at pH 7.4 was investigated, using isothermal titration calorimetry (ITC), UV and fluorescence emission spectroscopic methods. The results indicate that CeCl3, Ce3+, induces irreversible denaturation of the HSA structure. The UV absorption intensity of HSA + Ce3+ shows a slight blueshift in the absorbance wavelength with increasing Ce3+ concentration. The fluorescence intensity was increased regularly and a slight redshift was observed in the emission wavelength. The HSA + Ce3+ complex quenches the fluorescence of HSA and changes the microenvironment of tryptophan residue. The emission intensity increases suggesting the loss of the tertiary structure of HSA. The results obtained from the ITC data are in agreement with the spectroscopic methods. The strong negative cooperativity of Ce3+ binding with HSA (Table 1) recovered from the extended solvation model, indicates that HSA has been denatured as a result of its interaction with Ce3+ ions.

Fluorescence spectroscopic analysis on interaction of fleroxacin with pepsin

The interaction between fleroxacin (FLX) and pepsin was investigated by spectrofluorimetry. The effects of FLX on pepsin showed that the microenvironment of tryptophan residues and molecular conformation of pepsin were changed based on fluorescence quenching and synchronous fluorescence spectroscopy in combination with three-dimensional fluorescence spectroscopy. Static quenching was suggested and it was proved that the fluorescence quenching of pepsin by FLX was related to the formation of a new complex and a non-radiation energy transfer. The quenching constants KSV , binding constants K and binding sites n were calculated at different temperatures. The molecular interaction distance (r = 6.71) and energy transfer efficiency (E = 0.216) between pepsin and FLX were obtained according to the Forster mechanism of non-radiation energy transfer. Hydrophobic and electrostatic interaction played a major role in FLX-pepsin association. In addition, the hydrophobic interaction and binding free energy were further tested by molecular modeling study.

OmpC-like porin from Yersinia pseudotuberculosis: Molecular characteristics, physico-chemical and functional properties

Pore-forming protein from the outer membrane of Yersinia pseudotuberculosis cultured at 37°C has been isolated and characterized. Comparative analysis of the primary and three-dimensional structures of this protein and of OmpC porin from E. coli was carried out, functional properties of these proteins have been studied using bilayer lipid membranes (BLM) technique. The degree of homology, molecular mass and pore-forming properties of the isolated porin was found to be closer to those of OmpC porin from E. coli than OmpF porin from Y. pseudotuberculosis. The value of the most probable conductivity of OmpC porin from Y. pseudotuberculosis (0.18 pS) in BLM corresponded to the conductivity of the native trimer of this protein. Using CD spectroscopy, the porins investigated were shown to belong to the β-structured proteins. Data of the primary structure and intrinsic protein fluorescence revealed essential differences in localization and microenvironment of tryptophan residues in the porins investigated. Participation of external loops L2 and L6 in the formation of the antigenic structure of OmpC porin from Y. pseudotuberculosis was demonstrated. On the basis of crystal structure of osmoporin from Klebsiella pneumoniae, three-dimensional models of the monomer and trimer of the Y. pseudotuberculosis porin were obtained. Using Web server AGGRESCAN, the localization of protein structure sites with the increased aggregation capability (hot spots) has been deter-mined. It turned out that some of these zones localize in the region of intramonomeric contacts in the porin trimer; however, a large part of them is located on the external surface of the β-barrel. The process of thermal denaturation has been studied and the melting points of the porins were determined. It was found that significant changes in the microenvironment of the indole fluorophores (especially tryptophan residues of spectral class I) took place in the process of the thermodenaturation of the proteins. These changes preceded the irreversible conformational transition observed for the E. coli porin at 77°C and for the Y. pseudotuberculosis porin at 70°C.

Probing a family GH11 endo-β-1,4-xylanase inhibition mechanism by phenolic compounds: Role of functional phenolic groups

Abstract Phenolic compounds generated from lignin degradation during the pre-treatment step in the process of producing bioethanol from lignocellulosic biomass are known to be inhibitory to enzymatic hydrolysis and fermentation. The inactivation mechanism of a GH11 endoxylanase (Tx-Xyl) by several phenolic compounds varying in their hydroxyl and methoxyl radical content was investigated. Apparent kinetic inactivation parameters were measured as an approximate index of the inhibitory effects. All the tested aromatic compounds had strong negative impact on enzyme activity and kinetic analysis revealed non competitive multi-site inhibition mechanism. The interactions between Tx-Xyl and the phenolic compounds were further studied by steady-state (tryptophan) fluorescence spectroscopy. Changes in λ max of emission and quenching of fluorescence intensity indicated changes in the microenvironment of tryptophan residues. In agreement with the kinetic parameters, the fluorescence derived binding constants evidenced higher enzyme–phenolics interaction affinity with increasing phenolic hydroxyl radical content, suggesting clear correlations of such radicals with the inhibitory effects. Results indicated that the inhibitory effects of phenolic compounds on Tx-Xyl activity are most likely brought about by conformational alterations of the enzyme protein inducing steric inactivation.