Site IIA Research Articles

Neutralizing and Epitope-Specific Antibodies against Respiratory Syncytial Virus in Maternal and Cord Blood Paired Samples.

Only a few qualitative studies of neutralizing antibody titers (NATs) against respiratory syncytial virus (RSV) have focused on epitope-specific antibody (ESA) levels. Here, NATs against RSV in sera were measured using the blood of 412 mothers and cord blood (CB) of 95 of the 412 mother-child pairs. ESA levels against sites zero (Ø) and IIa of the F protein of RSV were measured in 87 of the 95 mother-child pairs. The median gestational age was 39 weeks. The NATs and ESA levels in CB were slightly higher than those in maternal blood (MB). The NATs for RSV subtype A (RSV-A) in MB and CB showed a positive correlation (r = 0.75). The ESA levels against sites Ø and IIa in MB and CB showed positive correlations, r = 0.76 and r = 0.69, respectively. In MB, the NATs and ESA levels against RSV were positively correlated, more significantly against site Ø (RSV-A: r = 0.70, RSV-B: r = 0.48) than against site IIa (RSV-A: r = 0.19, RSV-B: r = 0.31). Sufficient amounts of ESAs against sites Ø and IIa of RSV were transferred from mothers to term infants. ESA levels against site Ø contribute to NATs.

Evaluation of diflunisal a difluoro-derivative of salicylic acid as an inhibitor of human serum albumin using molecular docking tools

Aim: To evaluate the binding interaction of diflunisal at active site of Human Serum Albumin (HSA), an important enzyme responsible for osteoarthritis disease. Methodology: The gas phase molecule was optimized with B3LYP/6-311G** basis set, while a single point energy calculation was carried out for the molecule lifted from the active site. Bader’s theory of atoms in molecules (AIM) was used to determine the electron density and Laplacian of electron density. The protein was assigned with polar hydrogens and Kollman charges. Iterated local search procedure was performed during docking, during this, both protein and ligand were considered as rigid. Among the ten poses, lowest binding energy pose was considered for further ligand protein interaction analysis and QTAIM studies. Results: A close observation of the results shows that diflunisal has interactions in the binding sites IIA and IIIA of HSA as reported earlier. Two strong classical H-bonding interactions, three hydrophobic contacts with the amino acids VAL456, ALA194 andARG197, two close fluorine interactions, and two Pi-Sigma interactions have been observed. Apart from the H-bond interactions, the stability of HAS-Diflunisal complex was further empathized by five hyrdrophobic interactions framed between hydroxyl benzoic acid ring with ALA194, GLN459 and ARG197amino acid residue and difluorobenzene ring with ALA194 and VAL456. Interpretation: The results of drug-likeness study showed that diflunisal is highly reactive and less toxic.

Interactions between stipuol enantiomers and human serum albumin

Natural polyacetylenes occur in food and herbal plants, have a wide range of bioactivities, and are recognized as important nutraceuticals. Stipuol is a natural polyacetylene present in the edible plant Panax notoginseng. The present study was aimed to study interactions of rac-stipuol and its enantiomers with human serum albumin (HSA) using multi-spectroscopic, molecular modeling and microscale thermophoresis. Steady-state and time-resolved fluorescence spectra manifest that the fluorescence quenching mechanism is mainly static in type. The bindings of (S)-stipuol, (R)-stipuol, rac-stipuol lead to some microenvironmental and slight conformational changes of HSA. Competitive ligand displacement experiments and molecular modeling studies revealed that stipuol enantiomers bind to HSA at subdomain III (site IIA). The calculated values of Ka and Kd showed that (R)-stipuol had a stronger binding affinity than (S)-stipuol. The results are informative for use of stipuol as a nutraceutical to improve human health.

Study of β-lactam-based drug interaction with albumin protein using optical, sensing, and docking methods.

The quality and strength of drug and albumin interaction affecting the drug-free concentration and physiological activity are important issues in pharmacokinetic research. In the present study, not only did we evaluate the binding strength of ceftriaxone and ceftizoxime to bovine serum albumin (BSA), but we also investigated the kinetic and thermodynamic parameters including KD, KA, ΔS, and ΔH. We applied in vitro optical fluorescence spectroscopy and surface plasmon resonance (SPR) sensing approaches as well as molecular docking analyses. The kinetic and thermodynamic investigations were done using different concentrations of drugs at three temperatures. Thermodynamic parameters visibly demonstrated that the binding was an exothermic and spontaneous process. The obtained negative values of both enthalpy change (ΔH) and entropy change (ΔS) in fluorescence and SPR and also molecular docking investigations showed that the major binding force involved in the complexation of drugs to BSA was hydrogen bonding. Static quenching was the foremost fluorescence quenching mechanism between them. Furthermore, the results of ΔG and KD values proved that the interaction of ceftriaxone-BSA was stronger than ceftizoxime-BSA. Finally, molecular docking confirmed that the preferable binding sites of ceftizoxime and ceftriaxone were site IIA and site IB of albumin, respectively.

Binding affinity of p-PD-based schiff-bases towards human serum albumin; in-vitro and in-silico assessment

The Schiff-bases {N,N′-Bis(2-hydroxybenzylidene)-benzene-1,4-diamine (L1), N,N′-Bis(5‑bromo-2-hydroxybenzylidene) -benzene-1,4-diamine (L2), N,N′-Bis(2‑hydroxy-3‑methoxy-benzylidene)-benzene-1,4-diamine (L3), and N,N′-Bis(2-hydroxynaphthylidene)-benzene-1,4-diamine (L4)} have been synthesised by the reaction of 1,4-phenylenediamine (p-PD) with the necessary aldehydes and characterised by UV–Vis, FT-IR, 1H NMR, and mass spectroscopy. Their solubilities, at room temperature, were determined according to the USP and BP classification. Quantum chemical calculations of the compounds L1 to L4 have been carried out by DFT at the B3LYP/6–311++G(d,p) level; these show the results of the calculations to be in accordance with the experimental ones. A comparative analysis of the experimental and calculated vibrational frequencies was performed and significant bands were identified.The binding affinity between our Schiff-bases and human serum albumin (hsa) was studied under simulated physiological conditions, using absorbance titration experiments, fluorescence spectroscopy, circular dichroism (CD), and molecular docking (MD). Interaction results revealed one molecule of synthesised Schiff-bases to bind to the protein. MD results suggested that the binding site of L1 and L3 was site IA and that of L2 and L4 were sites IIIB and IIA, respectively. In vitro anticancer activity of the synthesised compounds was evaluated against the human hepatocellular carcinoma (HepG2) and human breast (MCF-7) cancer cells using MTT assays. Among the compounds, L4 (containing a naphthyl substituents) exhibited the highest anticancer activity with IC50 values of 12.90 ± 8.91 and 13.90 ± 7.88 against the HepG2 and MCF-7 cell lines after an incubation of 24 h.

Molecular mechanism and thermodynamic study of Rosuvastatin interaction with human serum albumin using a surface plasmon resonance method combined with a multi-spectroscopic, and molecular modeling approach

Rosuvastatin (ROS) is an anti-cholesterol drug belonging to statin drugs. A multi-spectroscopic approach combined with a molecular modeling technique was used to assess ROS association with human serum albumin (HSA). Besides, an HSA immobilized surface plasmon resonance (SPR) chip was used to obtain kinetic parameters (ka, kd, and KD). Fluorescence quenching titrations revealed that ROS interacts with HSA via a dynamic, exothermic, enthalpy-driven mechanism. Hydrogen bonds and van der Waals interactions as the most prevalent bonding forces contribute to ROS-HSA complex formation. ROS binding to HSA alters HSA conformation. The SPR results indicated that ROS and HSA have a strong interaction possessing an equilibrium constant (KD) of 1.55 × 10−8 M at 298 K. A competitive analysis of site markers showed that ROS has a higher tendency to bind to the warfarin binding site (site IIA), which may explain why warfarin has a higher anticoagulant effect in ROS users. FRET analysis indicated that non-radiation energy transfer occurred between ROS and HSA. According to molecular docking studies, ROS prefers binding sites IB and IIA while the ROS-HSA complex stabilizes due to the hydrogen bond and π-π interaction. The presence of hydrogen-bond donors and acceptors, as well as aromatic ROS moieties, facilitates such interactions.

Design and Synthesis of Novel Peptides to Protect Ferulic Acid against Ultraviolet Radiation Based on Domain Site IIA of Bovine Serum Albumin.

Ferulic acid (FA) is known for its excellent antioxidant properties, which can provide many health benefits. One of its drawbacks is its instability under UVA light, which limits its potency. In this study, the new peptides LW2 (QNKRFYFRKNQ) and CW2 (a cyclic form of LW2) were designed based on bovine serum albumin site IIA conformation. A UVA irradiation experiment was performed to investigate the protective ability of these peptides towards FA against UVA damage. The percentages of FA remaining under UV irradiation due to the protection of CW2 and LW2 were 83% and 76%, respectively. The results showed the importance of the cationic residues and hydrophobic residues included in the peptide sequences. Moreover, the cyclic rigid structure showed greater protective ability as compared to its linear counterpart.

Stabilization of the ruthenium (II) and -(III) centres by chelating N-donor ligands: Synthesis, characterization, biomolecular affinities and computational studies

Anticancer activities of ruthenium Schiff base metal complexes have been closely correlated to their steric factors and physicochemical properties. The core objectives of our research study were to synthesize and characterize new ruthenium Schiff base compounds derived from cinnamaldehyde, cuminaldehyde or 4-aminoantipyrine. In addition, the stereo-electronic features of the aforementioned metal compounds and two selected previously reported ruthenium Schiff base compounds were related to their antioxidant and biomolecular interaction capabilities. Therefore, we demonstrated the formation of novel mononuclear ruthenium(III) compounds with chelating N-donor ligands: fac-[RuCl3(PPh3)(ap)] (1) (ap = 4-aminoantipyrine), trans-P-[Ru(PPh3)2(cinap)2](PF6) (2) (cinap = 1,5-dimethyl-2-phenyl-4-{[3-phenylprop-2-en-1-ylidene]amino}-1,2-dihydro-3H-pyrazol-3-one) and cis-Cl, trans-P-[RuCl2(PPh3)2(cumbh)] (3) (cumbh = N'-(4-isopropylbenzylidene)benzohydrazide). Structural confirmations were conducted primarily by spectroscopic techniques while the single crystal X-ray analysis revealed the distorted octahedrons of the respective metal compounds. Voltammetry experiments of 1–3 illustrated one-electron quasi-reversible redox waves which are attributed to metal oxidation state interconversions. CT-DNA binding affinities and modes of the novel metal complexes 1–3 as well as the formerly published ruthenium Schiff base compounds, trans-P, cis-Cl-[Ru(pch)Cl2(PPh3)2] (4) (pch = 4-((pyridine-2yl-imino)methylene)-chromone) and cis-[RuCl2(bpap)(PPh3)] (5) (bpap = 2,6-bis-((antipyrine-imino)methylene)pyridine) were experimentally and computationally investigated. The paramagnetism of 1–4 and the proton-donor abilities of all the metal compounds promoted significantly higher NO and DPPH radical scavenging activities than the natural antioxidant, vitamin C. Experimental electronic BSA titrations revealed that the metal compounds are ideal binders which preferentially bind to site IIA. The X-ray and spectroscopic data were supported by the computed data which was simulated using the density functional theory method.

Interaction study of monoisoamyl dimercaptosuccinic acid with bovine serum albumin using biophysical and molecular docking approaches

Monoisoamyl 2,3-dimercaptosuccinic acid (MiADMSA), a lipophilic chelator has been evaluated for its potential use as an antidote in arsenic poisoning. The pharmacokinetics and pharmacodynamics properties of a drug could be understood via study its mechanism of interaction with bovine serum albumin protein (BSA). Therefore, the interaction between MiADMSA with BSA was investigated using various spectroscopic techniques and computational methods. Linear quenching of BSA intrinsic fluorescence intensity with the increasing concentration of MiADMSA was observed in the fluorescence study. Furthermore, synchronous results revealed that MiADMSA slightly changed the conformation of BSA. The binding constant value of the BSA-MiADMSA complex was found 1.60 × 104 M−1 at 298 K. The value of thermodynamic parameters ΔG, ΔH, and ΔS described that the process is spontaneous, endothermic, and hydrophobic forces are involved in the interaction of MiADMSA with BSA. Competitive site marker experiments showed that MiADMSA binds to site-II of BSA. Conformational changes of BSA with the interaction of MiADMSA were apparent by CD, UV–Visible, FT-IR, and 3D fluorescence spectroscopy. To strengthen the experimental findings we have also performed a theoretical study on the BSA-MiADMSA complex. Two sites were identified with docking score of − 6.642 kcal/mol at site IIa and − 3.80 kcal/mol for site IIb via molecular docking study. Molecular dynamics simulation study inferred the stability of the BSA-MiADMSA complex which was analyzed in a long simulation run. The experimental and computational studies have shown the effective binding of MiADMSA with BSA which is essential for the transportation and elimination of a drug from the body.

Modulation of excited-state photodynamics of ESIPT probe 1′-hydroxy-2′-acetonaphthone (HAN) on interaction with bovine serum albumin

The 1′-hydroxy-2′-acetonaphthone (HAN) is a well-known excited-state intramolecular proton-transfer (ESIPT) dye that has been used as a useful fluorescence probe to understand various homogenous and microheterogenous media. However, interaction of HAN with proteins is still largely less explored. The present study is aimed to investigate the interaction of HAN with the most abundant animal circulatory protein, bovine serum albumin (BSA), using multi-spectroscopic techniques and molecular docking studies. The photophysical properties of HAN undergo significant modulation upon its interaction with BSA protein. The confinement and reduced polarity experienced by bound HAN into BSA binding pocket causes a large fluorescence enhancement. The binding constant for HAN-BSA complex is evaluated to be ∼4.3 × 104 M−1. Time-resolved fluorescence measurements reveal the exclusive presence of only keto (K*) form and twisted keto rotamer (KR*) form of the dye in the excited state inside the BSA binding pocket, though in the ground state the dye exists entirely in its enol (E) form. Results suggest a quantitative ESIPT reaction for HAN even inside the BSA pocket. Time resolved fluorescence anisotropy studies indicate very tight binding of HAN into the BSA pocket such that independent rotational diffusion of the dye is completely ceased. Molecular docking studies provide best docking conformation and involved interaction forces, suggesting the preferred binding of HAN to the site IIA (Sudlow's site I) of the BSA protein. Observed photophysical results provide valuable insights of HAN–BSA binding interaction, revealing for the first time that the intriguing excited-state equilibrium between K* and KR* forms of HAN is largely shifted towards KR* form, as the dye binds to the BSA pocket. To the best of our knowledge such an intriguing modulation in the excited state prototautomeric conversion process has not been reported before for the studied dye in the presence of any other host. Results of the present study can be useful in understanding the pharmacology of structurally similar dyes/drugs, having relevance to drug delivery, pharmaceutical sciences, sensors, and so on.

Interactions of polyglycerol dendrimers with human serum albumin: insights from fluorescence spectroscopy and computational modeling analysis

Polyglycerol dendrimer synthesized from glycerol core (PGLyD) is an interesting reservoir macromolecule for the design of drug delivery systems due to their adequate blood biocompatibility. However, important features as the comprehension of the structural and dynamic characteristics and the interactions of PGLyD with blood proteins receptors remain unresolved. The high affinity and transport of HSA with drugs stimulated the docking simulations utilizing PGLyD as a ligand for the main HSA docking sites IIA and IIIA. HSA and the PGLyD structures were generated with the aid of Autodock Vina and the best conformations were determined by employing molecular docking. The molecular docking results indicate a thermodynamically favorable interaction suggesting a charge transfer complex formation between HSA and PGLyD. The interaction between PGLyD and HSA was investigated by fluorescence and the quenching mechanism of fluorescence of HSA by PGLyD was discussed. The binding constants and the number of binding sites were measured. The values of thermodynamic parameters ΔG, ΔH, and ΔS were calculated at three different temperatures. The experimental and computational results suggest that hydrophobic forces play a major role in stabilizing the HSA–PGLyD complex.

Tianeptine Esters Derivatives: A Study of Protein-Drug Interaction Performed by Fluorescence Quenching and Molecular Docking

The nature of binding between bovine serum albumin (BSA) and the antidepressant tianeptine and a new series of esters derivatives were studied in this paper. The interactions with BSA were investigated by UV-Vis and fluorescence spectroscopy at three different temperatures. The fluorescence quenching experiments showed that BSA interactions with tianeptine could be dynamic while to its esters a static mechanism was observed. The results showed that tianeptine quenches the intrinsic fluorescence of BSA more efficiently than its esters due to the presence of the free acid portion. The number of binding sites determined by fluorescence spectroscopy is approximately equal to 1 indicating that there is one binding site between BSA tianeptine esters, but the presence of a second interaction site for tianeptine at higher temperatures could be not ruled out. Molecular docking calculations point out a strong affinity of tianeptine and its esters to the site IIA of protein, supporting the hypothesis of a static quenching mechanism.

Molecular Interaction of Hemorrheologic Agent, Pentoxifylline with Bovine Serum Albumin: An Approach to Investigate the Drug Protein Interaction Using multispectroscopic, Voltammetry and Molecular Modelling Techniques

Abstract The interaction between pentoxifylline (PTX) and bovine serum albumin (BSA) was studied under physiological condition by spectroscopic, voltammetry and molecular modelling techniques. The results of fluorescence studies revealed that the quenching mechanism of BSA by PTX was a static procedure. Binding constant of PTX-BSA was calculated and its value found to be 8.895 × 104 M−1, which is in close agreement with the results obtained from UV-Visible and voltammetry approach. The negative values of thermodynamic parameters (ΔG, ΔH and ΔS) indicated that van der Waals force and hydrogen bonding play major roles in the interaction of PTX with BSA. Based on Forster’s theory of non-radiation energy transfer, the binding distance (r) between the donor (BSA) and acceptor (PTX) was found to be 5.38 nm (298 K). The results of UV-Visible and circular dichroism (CD) spectroscopy showed that PTX interacts with BSA and induces conformational changes by reducing the α-helix content. The results of the UV-visible and voltammetry studies were further verified by the molecular docking method. Molecular modelling studies revealed possible residues involved in the drug-protein interaction and indicated that PTX binds to Site IIA of BSA.

Interaction of tetramethyl-substituted BODIPY dye with bovine serum albumin: Spectroscopic study and molecular docking

In this paper the interaction between tetramethyl-substituted BODIPY and bovine serum albumin (BSA) was studied using a spectroscopic approach (UV/vis spectrophotometry, spectrofluorimetry) and molecular docking. The fluorescence of tetramethyl-substituted BODIPY was found to substantially quenched in the presence of BSA. It was observed that BODIPY interacts with the BSA in the sub-domain site IIA of BSA. The BODIPY-BSA complex is formed due to the prevailing specific interactions.

Hemagglutinin-specific neutralization of subacute sclerosing panencephalitis viruses.

Subacute sclerosing panencephalitis (SSPE) is a progressive, lethal complication of measles caused by particular mutants of measles virus (MeV) that persist in the brain despite high levels of neutralizing antibodies. We addressed the hypothesis that antigenic drift is involved in the pathogenetic mechanism of SSPE by analyzing antigenic alterations in the MeV envelope hemagglutinin protein (MeV-H) found in patients with SSPE in relation to major circulating MeV genotypes. To this aim, we obtained cDNA for the MeV-H gene from tissue taken at brain autopsy from 3 deceased persons with SSPE who had short (3–4 months, SMa79), average (3.5 years, SMa84), and long (18 years, SMa94) disease courses. Recombinant MeVs with a substituted MeV-H gene were generated by a reverse genetic system. Virus neutralization assays with a panel of anti-MeV-H murine monoclonal antibodies (mAbs) or vaccine-immunized mouse anti-MeV-H polyclonal sera were performed to determine the antigenic relatedness. Functional and receptor-binding analysis of the SSPE MeV-H showed activity in a SLAM/nectin-4–dependent manner. Similar to our panel of wild-type viruses, our SSPE viruses showed an altered antigenic profile. Genotypes A, G3, and F (SSPE case SMa79) were the exception, with an intact antigenic structure. Genotypes D7 and F (SSPE SMa79) showed enhanced neutralization by mAbs targeting antigenic site IIa. Genotypes H1 and the recently reported D4.2 were the most antigenically altered genotypes. Epitope mapping of neutralizing mAbs BH015 and BH130 reveal a new antigenic site on MeV-H, which we designated Φ for its intermediate position between previously defined antigenic sites Ia and Ib. We conclude that SSPE-causing viruses show similar antigenic properties to currently circulating MeV genotypes. The absence of a direct correlation between antigenic changes and predisposition of a certain genotype to cause SSPE does not lend support to the proposed antigenic drift as a pathogenetic mechanism in SSPE.

Measles Virus Hemagglutinin epitopes immunogenic in natural infection and vaccination are targeted by broad or genotype-specific neutralizing monoclonal antibodies

Measles virus (MV) remains a leading cause of vaccine-preventable deaths in children. Protection against MV is associated with neutralizing antibodies that preferentially recognize the viral hemagglutinin (MV-H), and to a lesser extent, the fusion protein (MV-F). Although MV is serologically monotypic, 24 genotypes have been identified. Here we report three neutralization epitopes conserved in the more prevalent circulating MV genotypes, two located in the MV-H receptor binding site (RBS) (antigenic site III) and a third in MV-H/MV-F interphase (antigenic site Ia) which are essential for MV multiplication. In contrast, two MV-H neutralization epitopes, showed a genotype-specific neutralization escape due to a single amino acid change, that we mapped in the “noose” antigenic site, or an enhanced neutralization epitope (antigenic site IIa). The monoclonal antibody (mAb) neutralization potency correlated with its binding affinity and was mainly driven by kinetic dissociation rate (koff). We developed an immunoassay for mAb binding to MV-H in its native hetero-oligomeric structure with MV-F on the surface of a MV productive steady-state persistently infected (p.i.) human cell lines, and a competitive-binding assay with serum from individuals with past infection by different MV genotypes. Binding assays revealed that a broad neutralization epitope, in RBS antigenic site, a genotype specific neutralization epitopes, in noose and IIa sites, were immunogenic in natural infection and vaccination and may elicit long-lasting humoral immunity that might contribute to explain MV immunogenic stability. These results support the design of improved measles vaccines, broad-spectrum prophylactic or therapeutic antibodies and MV-used in oncolytic therapies.

Multi-spectroscopic and molecular modeling studies of interaction between two different angiotensin I converting enzyme inhibitory peptides from gluten hydrolysate and human serum albumin

The present study was carried out to characterize Angiotensin-converting enzyme (ACE) inhibitory peptides which are released from the trypsin hydrolysate of wheat gluten protein. The binding of two inhibitory peptide (P4 and P6) to human serum albumin (HSA) under physiological conditions has been investigated by multi-spectroscopic in combination with molecular modeling techniques. Time-resolved and quenching fluorescence spectroscopies results revealed that the quenching of HSA fluorescence by P4 and P6 in the binary and ternary systems caused HSA-peptides complexes formation. The results indicated that both peptides quenched the fluorescence intensity of HSA through a static mechanism. The binding affinities and number of binding sites were obtained for the HSA-peptides complexes. The circular dichroism (CD) data revealed that the presence of both peptides increased the α-helix content of HSA and induced the remarkable folding of the polypeptide of the protein. Therefore, the CD data determined that the protein structure has been stabilized in the percent of ACE inhibitory peptides in binary and ternary systems. The binding distances between HSA and both peptides were estimated by the Forster theory, and it was revealed that nonradiative energy transfer from HSA to peptides occurred with a high probability. ITC experiments reveal that, in the absence and presence of P6, the dominant forces are electrostatic in binary and ternary systems. Furthermore, molecular modeling studies confirmed the experimental results. Molecular modeling investigation suggested that P4 bound to the site IA and IIA of HSA in binary and ternary systems, respectively. This study on the interaction of peptides with HSA should prove helpful for realizing the distribution and transportation of food compliments and drugs in vivo, elucidating the action mechanism and dynamics of food compliments and drugs at the molecular level. It should moreover be of great use for understanding the pharmacokinetic and pharmacodynamic mechanism of the food compliments and drugs.

Rabies virus isolates of India – Simultaneous existence of two distinct evolutionary lineages

Rabies is a fatal viral disease of serious public health implication. The disease is enzootic in India. In the present study, thirty six rabies virus isolates were obtained from terrestrial mammals of India during 2002–2012. Ecto-domain coding region of the glycoprotein gene from all the isolates were sequenced and the phylogenetic analysis was performed in relation to the global rabies and rabies related virus isolates. The Indian isolates grouped into two distinctly separate lineages with majority of the Indian isolates in Arctic like 1 lineage and the remaining isolates in sub-continental lineage. Isolates of the two distinct lineages were identified simultaneously from the same geographical region. Time scaled phylogenetic tree indicated that the sub-continental lineage of the virus is one of the earliest clade of rabies virus that diverged from bat rabies virus. On the contrary, the Arctic-like 1 lineage of India appeared to be a more recent divergence event. The amino acid sequence comparison revealed that all the major antigenic sites were almost conserved among the Indian isolates whereas few amino acid variations could be identified around site IIa, minor site I and IV. The dN/dS study based on G ecto-domain is in support of the earlier reports of strong purifying selection. In conclusion, it is evident that the Indian rabies virus isolates are of two major distinct lineages with distant phylogenetic and evolutionary relationship.

Determination of LMF Binding Site on a HSA-PPIX Complex in the Presence of Human Holo Transferrin from the Viewpoint of Drug Loading on Proteins

Holo transferrin (TF) and the natural complex of human serum albumin and protoporphyrin IX (HSA-PPIX) are two serum carrier proteins that can interact with each other. Such an interaction may alter their binding sites. In this study, fluorescence spectroscopy, as well as zeta potential and molecular modeling techniques, have been used to compare the complexes (HSA-PPIX)-LMF and [(HSA-PPIX)-TF]-LMF. The Ka1, Ka2, values of (HSA-PPIX)-LMF and [(HSA-PPIX)-TF]-LMF were 1.1×105 M−1, 9.7×106 M−1, and 2.0×104 M−1, 1.8×105 M−1, respectively, and the n1, n2 values were respectively 1.19, 1.53 and 1.17, 1.65. The second derivative of the Trp emission scan of (HSA-PPIX)-LMF exhibited one negative band at 310 nm, whereas for the [(HSA-PPIX)-TF]-LMF system, we observed one negative band at 316 nm indicating an increase in polarity around Trp. The effect of TF on the conformation of (HSA-PPIX)-TF was analyzed using three-dimensional fluorescence spectroscopy. The phase diagram indicated that the presence of a second binding site on HSA and TF was due to the existence of intermediate structures. Zeta potential analysis showed that the presence of TF increased the positive charges of the HSA-PPIX system. Site marker experiments revealed that the binding site of LMF to HSA-PPIX changed from Sudlow's site IIA to Sudlow's site IIIB in the presence of TF. Moreover, molecular modeling studies suggested the sub-domain IIIB in HSA as the candidate place for the formation of the binding site of LMF on the (HSA-PPIX)-TF complex.

A novel mechanism for antiglycative action of limonene through stabilization of protein conformation

Inhibition of protein glycation is known to ameliorate secondary complications in diabetes. In the present study antiglycative properties of limonene, a natural product, were evaluated using BSA as a model protein. AMG (aminoguanidine) was used as a positive control. Measurement of total AGEs (Advanced Glycation End-products) and specific AGEs revealed that limonene could inhibit protein glycation to the extent of 56.3% and 75.1% respectively at 50 μM concentration as against 54.4% and 82.2% by AMG at 1 mM. Congo red binding and CD (Circular Dichroism) analysis revealed inhibition of α-helix to β-sheet transition wherein 18.5% β-sheet structures were observed in glycated BSA (bovine serum albumin) as against 4.9% with limonene. Glycation of protein in the presence of urea was enhanced by 18%, while in the presence of limonene it was reduced by 23% revealing the stabilizing effect of limonene. Electrophoretic mobility was similar to the normal control and a zeta potential value of -12.1 mV as against -15.1 mV in diabetic control was observed. Inhibition of glycation in limonene treated samples was confirmed through LC-MS analysis wherein AGEs such as pentosidine, CML (N(ε)-(carboxymethyl)lysine), CEL (N(ε)-(carboxyethyl)lysine), MOLD (methylglyoxal-lysine dimer) and imidazolone observed in glycated samples were absent in limonene treated samples. PatchDock studies revealed that limonene could bind to the major glycation sites IB, IIA and IIB sub domains and AMG to the IIIA sub domain. Thus limonene is a potent protein glycation inhibitor that prevents protein glycation through a novel mechanism of stabilization of protein structure through hydrophobic interactions.