Spermine as a possible endogenous allosteric activator of carboxypeptidase A: multispectroscopic and molecular simulation studies

Abstract

Carboxypeptidase A (EC.3.4.17.1) is a zinc-containing proteolytic enzyme that removes the C-terminal amino acid from a peptide chain with the free carboxylate-terminal. In this study, the effect of spermine interaction on the structure and thermal stability of Carboxypeptidase A was investigated by ultraviolet???visible spectroscopy, fluorescence spectroscopy, circular dichroism, Kinetic measurement, molecular docking and simulation studies have also been followed at the pH of 7.5. The transition temperature of Carboxypeptidase A, as a criterion of protein thermal stability, in the presence of spermine was enhanced by increasing the concentration of spermine. The results of fluorescence intensity changes, at two temperatures of 308 and 318?K, also suggested that spermine had a great ability to quench the fluorescence of Carboxypeptidase A through the static quenching procedure. The thermodynamic parameters changes, including standard Gibbs free-energy, entropy and enthalpy, showed that the binding of spermine to Carboxypeptidase A was spontaneous and the hydrogen bonding and van der Waals interactions played a major role in stabilizing the Carboxypeptidase A–spermine complex. The changes in the content of the α-helix and the β-sheet of the Carboxypeptidase A with binding to spermine were shown by the CD spectra method. Further, kinetic studies revealed that by increasing concentration of spermine, the activity of Carboxypeptidase A was enhanced. Also, the docking study revealed that the hydrogen bonding and van der Waals interactions played a major role in stabilizing the Carboxypeptidase A–spermine complex. As a result, spermine could be considered as an activator and a stabilizer for Carboxypeptidase A.

Communicated by Ramaswamy H. Sarma     

Exploring the thermal stability and activity of α-chymotrypsin in the presence of spermine

Polyamines such as spermine can have interaction with protein. The aim of the present study was to investigate how spermine could influence the structure, thermal stability, and the activity of α-chymotrypsin. Kinetics, thermodynamics, molecular dynamics (MD), and docking simulations studies were conducted to investigate the effect of spermine on the activity and structure of α-Chymotrypsin (α-Chy) in 50 mM Tris–HCl buffer, with the pH 8, using different spectroscopic techniques as well as molecular docking and MD simulations. The stability and activity of α-Chy were increased in the presence of spermine. The results of the kinetic study showed that the activity of spermine was increased. Enzyme activation was accompanied by changes on the α-Chy conformation. Fluorescence intensity changes showed dynamic quenching during spermine binding. The fluorescence quenching of the α-Chy suggested the more polar location of Trp residues. Near-UV and Far-UV circular dichroism studies also demonstrated the transfer of Trp, Phe, and Tyr residues to a more flexible environment. The increase in the absorption of α-Chy in the presence of spermine was as a result of the formation of spermine–α-Chy complex. Molecular docking results revealed the presence of one binding site with a negative value for the Gibbs free energy of the binding of spermine to α-Chy. Docking study also revealed that van der Waals interactions and hydrogen bonds played a major role in stabilizing the complex.     

Investigation on the Interaction of Norgestrel with Human Serum Albumin Using Spectroscopy and Molecular‐Docking Method

The interaction of norgestrel with human serum albumin (HSA) was investigated by spectroscopy and molecular‐docking methods. Results of spectroscopy methods suggested that the quenching mechanism of norgestrel on HSA was static quenching and that the quenching process was spontaneous. Negative values of thermodynamic parameters (ΔG, ΔH, and ΔS) indicated that hydrogen bonding and van der Waals forces dominated the binding between norgestrel and HSA. Three‐dimensional fluorescence spectrum and circular dichroism spectrum showed that the HSA structure was slightly changed by norgestrel. Norgestrel mainly bound with Sudlow site I based on a probe study, as confirmed by molecular‐docking results. Competition among similar structures indicated that ethisterone and norethisterone affected the binding of norgestrel with HSA. CH₃ in R₁ had little effect on norgestrel binding with HSA. The surface hydrophobicity properties of HSA, investigated using 8‐anilino‐1‐naphthalenesulfonic acid, was changed with norgestrel addition.     

Interaction studies of aristolochic acid I with human serum albumin and the binding site of aristolochic acid I in subdomain IIA

Optical spectroscopy and molecular docking methods were used to examine the binding of aristolochic acid I (AAI) to human serum albumin (HSA) in this paper. By monitoring the intrinsic fluorescence of single Trp214 residue and performing displacement measurements, the specific binding of AAI in the vicinity of Sudlow's Site I of HSA has been clarified. An apparent distance of 2.53 nm between the Trp214 and AAI was obtained via fluorescence resonance energy transfer (FRET) method. In addition, the changes in the secondary structure of HSA after its complexation with the ligand were studied with circular dichroism (CD) spectroscopy, which indicated that AAI does not has remarkable effect on the structure of the protein. Moreover, thermal denaturation experiments clearly indicated that the HSA−AAI complexes are conformationally more stable. Finally, the binding details between AAI and HSA were further confirmed by molecular docking studies, which revealed that AAI was bound at subdomain IIA through multiple interactions, such as hydrophobic effect, van der Waals forces and hydrogen bonding.     

A Minimal or Concise Set of Definition of Life is Not Useful

The binding of ciprofloxacin to lysozyme in the presence of three Ag nano-particles of varying sizes was for the first time investigated by multispectroscopic and isothermal titration calorimetry techniques at pH 7.4. The results indicated that ciprofloxacin quenched the fluorescence intensity of lysozyme through a static mechanism but in the presence of size-II Ag nano-particles, there were two kinds of interaction behaviors. The interaction between ciprofloxacin and lysozyme occurred via a second type of binding site, whereas in the presence of the Ag nano-particles, some changes occurred. The secondary structure of lysozyme–ciprofloxacin in the presence of Ag nano-particles was determined by circular dichroism. The thermodynamic parameters of the interaction between ciprofloxacin and lysozyme in the presence of Ag nano-particles were measured according to the van’t Hoff equation. The enthalpy (ΔH^○) and entropy (ΔS^○) changes were calculated to be ?49.7 (kJ?mol^?1) and ?20.1 (J?mol^?1?K^?1), respectively, which indicated that the interaction of ciprofloxacin with lysozyme was driven mainly by van der Waals forces and hydrogen bonding. In the presence of the three different-sized Ag nano-particles, the enthalpic and the entropic changes were both negative which indicated that hydrogen bonding with van der Waals forces played major roles in the binding between ciprofloxacin and lysozyme. Recent developments in nano-materials offer new pathways for controlling the protein behavior through surface interactions. These data indicate that the recent research on nano-particle/protein interactions will emphasize the importance of such interactions in biological systems with applications including the diagnosis and treatment of human diseases.     

Molecular interaction of manganese based carbon monoxide releasing molecule (MnCORM) with human serum albumin (HSA)

In the present study, the interaction between the HSA and MnCORM in vitro under physiological conditions, was investigated through ultraviolet-visible (UV-vis) absorption, fluorescence, time-resolved fluorescence, circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopic techniques and in silico molecular docking methods. Binding parameters such as the binding constant, number of binding sites and binding force were obtained from the fluorescence data. Thermodynamic interaction revealed that the reaction was spontaneous (ΔG < 0) and hydrogen bond and van der Waals interaction were primarily involved in the binding. The changes induced in the secondary structure conformation due to the MnCORM interaction were monitored using CD and FT-IR spectroscopic techniques. The results showed reduction in α-helix conformation and corresponding increase in β-sheet and unordered structures due to slight unfolding. The time-resolved fluorescence decay confirmed the static quenching mechanism of the MnCORM. The molecular docking studies revealed that the MnCORM interacted at Sudlow’s site II of domain IIIA through hydrogen bond and van der Waals interactions. In order to understand the drug distribution and elimination, studies on the drug molecule interaction with HSA are vital. Therefore, it is evident that MnCORM interacts with HSA through ground state complex formation and thus suitable for in vivo delivery.     

The study on interactions between levofloxacin and model proteins by using multi-spectroscopic and molecular docking methods

The interactions of levofloxacin (LEV) with lysozyme (LYZ), trypsin and bovine hemoglobin (BHb) were investigated, respectively, by using multi-spectral techniques and molecular docking in vitro. Fluorescence studies showed that LEV quenched LYZ/trypsin fluorescence in a combined quenching ways and BHb fluorescence in a static quenching with binding constants of .14, .51 and .20 × 10⁵ L mol^?1 at 298 K, respectively. The thermodynamic parameters demonstrated that hydrophobic forces, hydrogen bonds, and van der Waals forces played the major role in the binding process. The binding distances between LEV and the inner tryptophan residues of LYZ, trypsin, and BHb were calculated to be 4.04, 3.38, and 4.52 nm, respectively. Furthermore, the results of circular dichroism spectra (CD), UV–vis, and three-dimensional fluorescence spectra indicated that the secondary structures of LYZ, trypsin, and BHb were partially changed by LEV with the α-helix percentage of LYZ-LEV system increased while that of BHb-LEV system was decreased, the β-sheet percentage of trypsin-LEV system increased from 41.3 to 42.9%. UV–vis spectral results showed that the binding interactions could cause conformational and some micro-environmental changes of LYZ, trypsin, and BHb. The results of molecular docking revealed that in LYZ and trypsin systems, LEV bound to the active sites residues GLU 35 and ASP 52 of LYZ and trypsin at the active site SER 195, and in BHb system, LEV was located in the central cavity, which was consistent with the results of synchronous fluorescence experiment. Besides, LEV made the activity of LYZ decrease while the activity of trypsin increased.     

Supramolecular interaction of 6-shogaol,a therapeutic agent of Zingiber officinale with human serum albumin as elucidated by spectroscopic,calorimetric and molecular docking methods

Background6-Shogaol, one of the main bioactive constituents of Zingiber officinale has been shown to possess various therapeutic properties. Interaction of a therapeutic compound with plasma proteins greatly affects its pharmacokinetic and pharmacodynamic properties.PurposeThe present investigation was undertaken to characterize the interaction between 6-shogaol and the main in vivo transporter, human serum albumin (HSA).MethodsVarious binding characteristics of 6-shogaol–HSA interaction were studied using fluorescence spectroscopy. Thermal stability of 6-shogaol–HSA system was determined by circular dichroism (CD) and differential scanning calorimetric (DSC) techniques. Identification of the 6-shogaol binding site on HSA was made by competitive drug displacement and molecular docking experiments.ResultsFluorescence quench titration results revealed the association constant, K_a of 6-shogaol–HSA interaction as 6.29 ± 0.33 × 10⁴ M⁻¹ at 25 ºC. Values of the enthalpy change (−11.76 kJ mol⁻¹) and the entropy change (52.52 J mol⁻¹ K⁻¹), obtained for the binding reaction suggested involvement of hydrophobic and van der Waals forces along with hydrogen bonds in the complex formation. Higher thermal stability of HSA was noticed in the presence of 6-shogaol, as revealed by DSC and thermal denaturation profiles. Competitive ligand displacement experiments along with molecular docking results suggested the binding preference of 6-shogaol for Sudlow's site I of HSA.ConclusionAll these results suggest that 6-shogaol binds to Sudlow's site I of HSA through moderate binding affinity and involves hydrophobic and van der Waals forces along with hydrogen bonds.     

Spectroscopic and molecular docking studies on interaction of two Schiff base complexes with bovine serum albumin

Abstract

This study was designed to examine interaction of two ternary copper (II) Schiff base complexes with bovine serum albumin (BSA), using spectroscopic and molecular docking techniques. The fluorescence quenching measurements revealed that the quenching mechanism was static and the binding site of both Schiff bases to BSA was singular. Förster energy transfer measurements, synchronous fluorescence spectroscopy, and docking study showed both Schiff bases bind to the Trp residues of BSA in short distances. Docking study showed that both Schiff base molecules bind with BSA by forming several hydrogen and van der Waals bonds. In addition, molecular docking study indicated that Schiff base A and Schiff base B were located within the binding pocket of subdomain IB and subdomain IIA of BSA, respectively. Results of Fourier transform-infrared spectroscopy demonstrated that bovine serum albumin interacts with both Schiff bases and the secondary structure of BSA was changed.

Communicated by Ramaswamy H. Sarma     

Probing the binding of two fluoroquinolones to lysozyme: a combined spectroscopic and docking study

Ciprofloxacin (CPFX) and enrofloxacin (ENFX) are two of the most widely used fluoroquinolones (FQs) in human and veterinary medicines. Their occurrence in the environment has received much attention because of the potential adverse effects on humans and ecosystem functions. In this paper, we investigated the interaction mechanism between the two FQs and lysozyme by the spectroscopic and molecular docking methods. As shown by the fluorescence spectroscopy, additions of CPFX or ENFX effectively quenched the intrinsic fluorescence of lysozyme, which was attributed to the formation of a moderately strong complex. The enthalpy change (ΔH) and entropy change (ΔS) indicated that van der Waals forces and hydrogen bonds were the dominant intermolecular forces in the binding of two FQs to lysozyme. Furthermore, data obtained by UV-vis absorption, synchronous fluorescence and circular dichroism (CD) suggested that both CPFX and ENFX could lead to the conformational and some microenvironmental changes of lysozyme. Finally, the molecular docking illustrated that the two FQs had specific interactions with the residues of Trp62 and Trp63.     

Exploring the interaction between tyrphostin 9 and human serum albumin using biophysical and computational methods

Abstract

Tyrphostin 9 (Tyr 9) is a potent platelet-derived growth factor receptor (PDGFR) inhibitor, which induces apoptosis in various cancer cell types. The binding of Tyr 9 to the major transport protein, human serum albumin (HSA) was investigated using several spectroscopic techniques and molecular docking method. Fluorescence quenching titration results showed progressive decrease in the protein fluorescence with increasing drug concentrations. A decreasing trend of the Stern-Volmer constant, K _sv with increasing temperature characterized the drug-induced quenching as static quenching, thus pointed towards the formation of Tyr 9–HSA complex. The binding constant of Tyr 9–HSA interaction was found to lie within the range 3.48–1.69?×?10⁵ M^?1 at three different temperatures, i.e. 15 °C, 25 °C and 35?°C, respectively and suggested intermediate binding affinity between Tyr 9 and HSA. The drug–HSA complex seems to be stabilized by hydrophobic forces, van der Waals forces and hydrogen bonds, as suggested from the thermodynamic data as well as molecular docking results. The far-UV and the near-UV CD spectral results showed slight alteration in the secondary and tertiary structures, respectively, of the protein upon Tyr 9 binding. Interaction of Tyr 9 with HSA also produced microenvironmental perturbations around protein fluorophores, as evident from the three-dimensional fluorescence spectral results but increased protein’s thermal stability. Both competitive drug binding results and molecular docking analysis suggested Sudlow’s Site I of HSA as the preferred Tyr 9 binding site.

Communicated by Ramaswamy H. Sarma     

Investigation on the interaction between myricetin and dihydromyricetin with trypsin, α-chymotrypsin,lysozyme by spectroscopy and molecular docking methods

The interaction between myricetin and dihydromyricetin with trypsin, α-chymotrypsin and lysozyme was investigated using multispectral and molecular docking methods. The results of fluorescence quenching revealed that myricetin and dihydromyricetin could quench the intrinsic fluorescence of three different proteinases through a static quenching procedure. The binding constant and number of binding sites at different temperatures were measured. The thermodynamic parameters obtained at different temperatures showed van der Waals interactions and hydrogen bonds played the main roles in the interaction of myricetin with trypsin and lysozyme, hydrophobic force was dominant both in myricetin with α-chymotrypsin interaction and dihydromyricetin with trypsin and lysozyme interaction, as for the electrostatic forces, it was mainly the driving force in dihydromyricetin binding to α-chymotrypsin. There was non-radiative energy transfer between three proteinases and myricetin or dihydromyricetin with high probability. The microenvironment of trypsin, α-chymotrypsin and lysozyme is changed. The docking studies revealed that myricetin and dihydromyricetin entered the hydrophobic cavity of three proteinases and formed hydrogen bonds. The binding affinity of myricetin or dihydromyricetin is different with the trypsin, α-chymotrypsin and lysozyme due to the different molecular structure.     

Structural reorganization of molecular sheets derived from cellulose II by molecular dynamics simulations

We investigated structural reorganization of two different kinds of molecular sheets derived from the cellulose II crystal using molecular dynamics (MD) simulations, in order to identify the initial structure of the cellulose crystal in the course of its regeneration process from solution. After a one-nanosecond simulation, the molecular sheet formed by van der Waals forces along the () crystal plane did not change its structure in an aqueous environment, while the other one formed by hydrogen bonds along the (1 1 0) crystal plane changed into a van der Waals-associated molecular sheet, such as the former. The two structures that were calculated showed substantial similarities such as the high occupancy of intramolecular hydrogen bonds between O3^H and O5 of over 0.75, few intermolecular hydrogen bonds, and the high occurrence of hydrogen bonding with water. The convergence of the two structures into one denotes that the van der Waals-associated molecular sheet can be the initial structure of the cellulose crystal formed in solution. The main chain conformations were almost the same as those in the cellulose II crystal except for a −16° shift of φ (dihedral angle of O5-C1-O1-C4) and the gauche-gauche conformation of the hydroxymethyl side group appears probably due to its hydrogen bonding with water. These results suggest that the van der Waals-associated molecular sheet becomes stable in an aqueous environment with its hydrophobic inside and hydrophilic periphery. Contrary to this, a benzene environment preferred a hydrogen-bonded molecular sheet, which is expected to be the initial structure formed in benzene.     

Hydrogen bonding-assisted interaction between amitriptyline hydrochloride and hemoglobin: spectroscopic and molecular dynamics studies

Herein, we have explored the interaction between amitriptyline hydrochloride (AMT) and hemoglobin (Hb), using steady-state and time-resolved fluorescence spectroscopy, UV–visible spectroscopy, and circular dichroism spectroscopy, in combination with molecular docking and molecular dynamic (MD) simulation methods. The steady-state fluorescence reveals the static quenching mechanism in the interaction system, which was further confirmed by UV–visible and time-resolved fluorescence spectroscopy. The binding constant, number of binding sites, and thermodynamic parameters viz. ΔG, ΔH, ΔS are also considered; result confirms that the binding of the AMT with Hb is a spontaneous process, involving hydrogen bonding and van der Waals interactions with a single binding site, as also confirmed by molecular docking study. Synchronous fluorescence, CD data, and MD simulation results contribute toward understanding the effect of AMT on Hb to interpret the conformational change in Hb upon binding in aqueous solution.     

Discovery of promising FtsZ inhibitors by E-pharmacophore, 3D-QSAR,molecular docking study,and molecular dynamics simulation

Filamentous temperature-sensitive protein Z (FtsZ), playing a key role in bacterial cell division, is regarded as a promising target for the design of antimicrobial agent. This study is looking for potential high-efficiency FtsZ inhibitors. Ligand-based pharmacophore and E-pharmacophore, virtual screening and molecular docking were used to detect promising FtsZ inhibitors, and molecular dynamics simulation was used to study the stability of protein-ligand complexes in this paper. Sixty-three inhibitors from published literatures with pIC₅₀ ranging from 2.483 to 5.678 were collected to develop ligand-based pharmacophore model. 4DXD bound with 9PC was selected to develop the E-pharmacophore model. The pharmacophore models validated by test set method and decoy set were employed for virtual screening to exclude inactive compounds against ZINC database. After molecular docking, ADME analysis, IFD docking and MM-GBSA, 8 hits were identified as potent FtsZ inhibitors. A 50?ns molecular dynamics simulation was implemented on the compounds to assess the stability between potent inhibitors and FtsZ. The results indicated that the candidate compounds had a high docking score and were strongly combined with FtsZ by forming hydrogen bonding interactions with key amino acid residues, and van der Waals forces and hydrophobic interactions had significant contribution to the stability of the binding. Molecular dynamics simulation results showed that the protein-ligand compounds performed well in both the stability and flexibility of the simulation process.     

Probing the combination of erlotinib hydrochloride,an anticancer drug,and human serum albumin: Spectroscopic,molecular docking,and molecular dynamic analyses

Human serum albumin (HSA) is a globular and monomeric protein in plasma that transports many drugs and compounds. Binding of some drugs to HSA can lead to changes in its stability and biological function. We investigated the binding interactions between erlotinib hydrochloride (Erlo) and HSA. Erlo is used to treat lung, pancreatic, and some other cancers. Experimental data showed that the fluorescence emission of the protein was quenched by Erlo using a static quenching mechanism. The calculation of the binding constant, K_b (1.57 × 10⁵ M⁻¹ at 300 K), confirmed the existence of a moderate binding interaction between Erlo and HSA. The interaction was enthalpy driven, spontaneous, and exothermic. The calculated thermodynamic parameters in agreement with simulation and molecular docking data showed that van der Waals and hydrogen bond forces played an important role in the interaction process. Molecular docking results indicated that Erlo has more affinity to bind to subdomain IIA (site I) of HSA. Molecular dynamics simulation analysis showed that the protein is stable in the presence of Erlo under simulation conditions.     

The identification of active compounds in Ganoderma lucidum var. antler extract inhibiting dengue virus serine protease and its computational studies

Abstract

The number of global dengue incidences is alarmingly high in recent years. The global distribution of four dengue serotypes has also added economic burden in the dengue-endemic countries. To discover the potent dengue virus inhibitors in the antler form of Ganoderma lucidum (Lingzhi or Reishi), the water extraction of normal G. lucidum and its antler form were conducted and the chemical compounds were identified by LC-MS. Six distinct chemical compounds identified in high abundance were hesperetin, thymidine, lucidenic acid, 11-aminoundecanoic acid, 5-carboxyvanillic acid and ganocin B. The water extracts of G. lucidum in its antler form inhibited the DENV2 NS2B-NS3 protease activity at 84.6?±?0.7%, higher than the normal G. lucidum. Then, molecular docking was performed on the homology model built from an in-house sequence. Docking simulation results showed that hesperetin and ganocin B were the best leads to bind at the catalytic triad of DENV2 NS2B-NS3pro via hydrogen bonding, van der Waals and pi-pi interactions. Extensive overlapping of HOMO-LUMO orbitals at the ringed regions of hesperetin helped to facilitate the entry of ligand to the catalytic triad cleft. LC-MS, molecular docking and density functional theory analyses confirmed that hesperetin was the strongest inhibitor against NS2B-NS3 protease.

Communicated by Ramaswamy H. Sarma     

Binding properties of palmatine to DNA: spectroscopic and molecular modeling investigations

Palmatine, an isoquinoline alkaloid, is an important medicinal herbal extract with diverse pharmacological and biological properties. In this work, spectroscopic and molecular modeling approaches were employed to reveal the interaction between palmatine and DNA isolated from herring sperm. The absorption spectra and iodide quenching results indicated that groove binding was the main binding mode of palmatine to DNA. Fluorescence studies indicated that the binding constant (K) of palmatine and DNA was ~ 10⁴ L·mol^?1. The associated thermodynamic parameters, ΔG, ΔH, and ΔS, indicated that hydrogen bonds and van der Waals forces played major roles in the interaction. The effects of chemical denaturant, thermal denaturation and pH on the interaction were investigated and provided further support for the groove binding mode. In addition to experimental approaches, molecular modeling was conducted to verify binding pattern of palmatine–DNA. Copyright © 2015 John Wiley & Sons, Ltd.     

Protein-RNA interactions: a structural analysis

A detailed computational analysis of 32 protein–RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein–double-stranded DNA and protein–single-stranded DNA complexes. The interface properties of the protein–RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein–RNA and protein–DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein–RNA complexes, backbone contacts were more dominant in the protein–DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level.