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1.  Nanoparticles in relation to peptide and protein aggregation 
Over the past two decades, there has been considerable research interest in the use of nanoparticles in the study of protein and peptide aggregation, and of amyloid-related diseases. The influence of nanoparticles on amyloid formation yields great interest due to its small size and high surface area-to-volume ratio. Targeting nucleation kinetics by nanoparticles is one of the most searched for ways to control or induce this phenomenon. The observed effect of nanoparticles on the nucleation phase is determined by particle composition, as well as the amount and nature of the particle’s surface. Various thermodynamic parameters influence the interaction of proteins and nanoparticles in the solution, and regulate the protein assembly into fibrils, as well as the disaggregation of preformed fibrils. Metals, organic particles, inorganic particles, amino acids, peptides, proteins, and so on are more suitable candidates for nanoparticle formulation. In the present review, we attempt to explore the effects of nanoparticles on protein and peptide fibrillation processes from both perspectives (ie, as inducers and inhibitors on nucleation kinetics and in the disaggregation of preformed fibrils). Their formulation and characterization by different techniques have been also addressed, along with their toxicological effects, both in vivo and in vitro.
PMCID: PMC3928455  PMID: 24611007
amyloid formation; inducer; inhibitor; nanoparticle; nucleation; toxicity
2.  Flower-shaped ZnO nanoparticles synthesized by a novel approach at near-room temperatures with antibacterial and antifungal properties 
Due to enormous applications of metal oxide nanoparticles in research and health-related applications, metal oxide nanoparticles are increasingly being developed through cheaper and more user-friendly approaches. We have formulated a simple route to synthesize zinc oxide nanoparticles (ZNPs) by a sol–gel method at near-room temperatures 25°C, 35°C, 55°C, and 75°C. The results are analyzed by X-ray diffraction, scanning electron microscopy with energy-dispersive X-ray spectroscopy, and ultraviolet-visible absorption spectroscopy. The effect of different temperature conditions (25°C–75°C) on the particulate sizes (23.7–88.8 nm), pH levels (11.7–11.9), and morphologies (slender needle–broad arrow) of flower-shaped ZNP colonies is studied. A possible mechanism depicting the growth rates at different temperatures and of different facets, mainly towards the <0 0 0 I> and <0 I Ī 0> planes of the ZNPs has also been discussed. The values of λmax (293–298 nm) suggest that ZNPs prepared at 55°C are the most effective ultraviolet B absorbers, and that they can be used in sunscreens. Highly significant antimicrobial activity against medically important Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria and fungi (Candida albicans) by these ZNPs was also revealed. As S. aureus and C. albicans are responsible for many contagious dermal infections such as abscesses, furuncles, carbuncles, cellulitis, and candidiasis, we can postulate that our fabricated ZNPs may be useful as antimicrobial agents in antiseptic creams and lotions for the treatment of skin diseases.
PMCID: PMC3925235  PMID: 24550675
antimicrobial activity; cetyl trimethyl ammonium bromide; flower-shape zinc oxide nanoparticles; near-room temperature; sol–gel method; skin disease
3.  A Comprehensive Insight into Binding of Hippuric Acid to Human Serum Albumin: A Study to Uncover Its Impaired Elimination through Hemodialysis 
PLoS ONE  2013;8(8):e71422.
Binding of hippuric acid (HA), a uremic toxin, with human serum albumin (HSA) has been examined by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), molecular docking, circular dichroism (CD) and fluorescence spectroscopy to understand the reason that govern its impaired elimination through hemodialysis. ITC results shows that the HA binds with HSA at high (Kb ∼104) and low affinity (Kb ∼103) sites whereas spectroscopic results predict binding at a single site (Kb∼103). The HA form complex with HSA that involves electrostatic, hydrogen and hydrophobic binding forces as illustrated by calculated thermodynamic parameters. Molecular docking and displacement studies collectively revealed that HA bound to both site I and site II; however, relatively strongly to the later. Esterase-like activity of HSA confirms the involvement of Arg410 and Tyr411 of Sudlow site II in binding of HA. CD results show slight conformational changes occurs in the protein upon ligation that may be responsible for the discrepancy in van’t Hoff and calorimetric enthalpy change. Furthermore, an increase in and is observed from DSC results that indicate increase in stability of HSA upon binding to HA. The combined results provide that HA binds to HSA and thus its elimination is hindered.
PMCID: PMC3739763  PMID: 23951159
4.  Flexibility of EF-hand motifs: structural and thermodynamic studies of Calcium Binding Protein-1 from Entamoeba histolytica with Pb2+, Ba2+, and Sr2+ 
BMC Biophysics  2012;5:15.
EF-hand proteins can be activated by the binding of various heavy metals other than calcium, and such complexes can disturb the calcium-signaling pathway and cause toxicity and disease causing state. So far, no comprehensive study has been done to understand different heavy metals binding to calcium signaling proteins.
In this work, the flexibility of the EF-hand motifs are examined by crystallographic and thermodynamic studies of binding of Pb2+, Ba2+ and Sr2+ to Calcium Binding Protein-1 from Entamoeba histolytica (EhCaBP1). The structures of the EhCaBP1- heavy metal complexes are found to be overall similar, nevertheless specific differences in metal coordination, and small differences in the coordination distances between the metal and the ligands in the metal binding loop. The largest such distances occur for the Ba2+- EhCaBP1 complex, where two bariums are bound with partial occupancy at the EF2 motif. Thermodynamic studies confirm that EhCaBP1 has five binding sites for Ba2+ compared to four binding sites for the other metals. These structures and thermodynamic studies reveal that the EF-hand motifs can accommodate several heavy atoms with similar binding affinities. The binding of Ca2+ to the 1st, 2nd and 4th sites and the binding of Ba2+ to the 1st, 2nd, 4th and 5th sites are both enthalpically and entropically driven, whereas the binding of Sr2+ to the 1st, 2nd and 4th sites are simply enthalpy driven, interestingly in agreement with ITC data, Sr2+ do not coordinate with water in this structure. For all the metals, binding to the 3rd site is only entropy driven.
Energetically, Ca2+ is preferred in three sites, while in one site Ba2+ has better binding energy. The Sr2+-coordination in the EF hand motifs is similar to that of the native Ca2+ bound structure, except for the lack of water coordination. Sr2+ coordination seems to be a pre-formed in nature since all seven coordinating atoms are from the protein itself, which also correlates with entropy contributions in Sr2+ binding. These findings improve our understanding of metal association with calcium binding proteins and of metal induced conformational changes.
PMCID: PMC3483242  PMID: 22906057
Calcium sensor; Calcium binding protein; Coordination geometry; EF-hand motifs; Anthropogenic toxicant; Domain swapped manner; Anomalous signal
5.  Correction: In Silico Prediction and Analysis of Caenorhabditis EF-hand Containing Proteins 
PLoS ONE  2012;7(8):10.1371/annotation/f7f4b3be-c835-440b-a851-7eb62c142260.
PMCID: PMC3935724
6.  Pollutant-Induced Modulation in Conformation and β-Lactamase Activity of Human Serum Albumin 
PLoS ONE  2012;7(6):e38372.
Structural changes in human serum albumin (HSA) induced by the pollutants 1-naphthol, 2-naphthol and 8-quinolinol were analyzed by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The alteration in protein conformational stability was determined by helical content induction (from 55 to 75%) upon protein-pollutant interactions. Domain plasticity is responsible for the temperature-mediated unfolding of HSA. These findings were compared to HSA-hydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators. Pollutants cause conformational changes and catalytic modifications of the protein (increase in β-lactamase activity from 100 to 200%). HSA-pollutant interactions mediate other protein-ligand interactions, such as HSA-nitrocefin. Therefore, this protein can exist in different conformations with different catalytic properties depending on activator binding. This is the first report to demonstrate the catalytic allostericity of HSA through a mechanistic approach. We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.
PMCID: PMC3369883  PMID: 22685563
7.  In Silico Prediction and Analysis of Caenorhabditis EF-hand Containing Proteins 
PLoS ONE  2012;7(5):e36770.
Calcium (Ca+2) is a ubiquitous messenger in eukaryotes including Caenorhabditis. Ca+2-mediated signalling processes are usually carried out through well characterized proteins like calmodulin (CaM) and other Ca+2 binding proteins (CaBP). These proteins interact with different targets and activate it by bringing conformational changes. Majority of the EF-hand proteins in Caenorhabditis contain Ca+2 binding motifs. Here, we have performed homology modelling of CaM-like proteins using the crystal structure of Drosophila melanogaster CaM as a template. Molecular docking was applied to explore the binding mechanism of CaM-like proteins and IQ1 motif which is a ∼25 residues and conform to the consensus sequence (I, L, V)QXXXRXXXX(R,K) to serve as a binding site for different EF hand proteins. We made an attempt to identify all the EF-hand (a helix-loop-helix structure characterized by a 12 residues loop sequence involved in metal coordination) containing proteins and their Ca+2 binding affinity in Caenorhabditis by analysing the complete genome sequence. Docking studies revealed that F165, F169, L29, E33, F44, L57, M61, M96, M97, M108, G65, V115, F93, N104, E144 of CaM-like protein is involved in the interaction with IQ1 motif. A maximum of 170 EF-hand proteins and 39 non-EF-hand proteins with Ca+2/metal binding motif were identified. Diverse proteins including enzyme, transcription, translation and large number of unknown proteins have one or more putative EF-hands. Phylogenetic analysis revealed seven major classes/groups that contain some families of proteins. Various domains that we identified in the EF-hand proteins (uncharacterized) would help in elucidating their functions. It is the first report of its kind where calcium binding loop sequences of EF-hand proteins were analyzed to decipher their calcium affinities. Variation in Ca+2-binding affinity of EF-hand CaBP could be further used to study the behaviour of these proteins. Our analyses postulated that Ca+2 is likely to be key player in Caenorhabditis cell signalling.
PMCID: PMC3360750  PMID: 22701514
8.  SDS Can Be Utilized as an Amyloid Inducer: A Case Study on Diverse Proteins 
PLoS ONE  2012;7(1):e29694.
Sodium dodecyl sulphate (SDS), an anionic surfactant that mimics some characteristics of biological membrane has also been found to induce aggregation in proteins. The present study was carried out on 25 diverse proteins using circular dichroism, fluorescence spectroscopy, dye binding assay and electron microscopy. It was found that an appropriate molar ratio of protein to SDS readily induced amyloid formation in all proteins at a pH below two units of their respective isoelectric points (pI) while no aggregation was observed at a pH above two units of pI. We also observed that electrostatic interactions play a leading role in the induction of amyloid. This study can be used to design or hypothesize a molecule or drug, which may counter act the factor responsible for amyloid formation.
PMCID: PMC3257246  PMID: 22253760
9.  Stereo-Selectivity of Human Serum Albumin to Enantiomeric and Isoelectronic Pollutants Dissected by Spectroscopy, Calorimetry and Bioinformatics 
PLoS ONE  2011;6(11):e26186.
1–naphthol (1N), 2–naphthol (2N) and 8–quinolinol (8H) are general water pollutants. 1N and 2N are the configurational enantiomers and 8H is isoelectronic to 1N and 2N. These pollutants when ingested are transported in the blood by proteins like human serum albumin (HSA). Binding of these pollutants to HSA has been explored to elucidate the specific selectivity of molecular recognition by this multiligand binding protein. The association constants (Kb) of these pollutants to HSA were moderate (104–105 M−1). The proximity of the ligands to HSA is also revealed by their average binding distance, r, which is estimated to be in the range of 4.39–5.37 nm. The binding free energy (ΔG) in each case remains effectively the same for each site because of enthalpy–entropy compensation (EEC). The difference observed between ΔCpexp and ΔCpcalc are suggested to be caused by binding–induced flexibility changes in the HSA. Efforts are also made to elaborate the differences observed in binding isotherms obtained through multiple approaches of calorimetry, spectroscopy and bioinformatics. We suggest that difference in dissociation constants of pollutants by calorimetry, spectroscopic and computational approaches could correspond to occurrence of different set of populations of pollutants having different molecular characteristics in ground state and excited state. Furthermore, our observation of enhanced binding of pollutants (2N and 8H) in the presence of hemin signifies that ligands like hemin may enhance the storage period of these pollutants in blood that may even facilitate the ill effects of these pollutants.
PMCID: PMC3206814  PMID: 22073150
10.  Elimination of Endogenous Toxin, Creatinine from Blood Plasma Depends on Albumin Conformation: Site Specific Uremic Toxicity & Impaired Drug Binding 
PLoS ONE  2011;6(2):e17230.
Uremic syndrome results from malfunctioning of various organ systems due to the retention of uremic toxins which, under normal conditions, would be excreted into the urine and/or metabolized by the kidneys. The aim of this study was to elucidate the mechanisms underlying the renal elimination of uremic toxin creatinine that accumulate in chronic renal failure. Quantitative investigation of the plausible correlations was performed by spectroscopy, calorimetry, molecular docking and accessibility of surface area. Alkalinization of normal plasma from pH 7.0 to 9.0 modifies the distribution of toxin in the body and therefore may affect both the accumulation and the rate of toxin elimination. The ligand loading of HSA with uremic toxin predicts several key side chain interactions of site I that presumably have the potential to impact the specificity and impaired drug binding. These findings provide useful information for elucidating the complicated mechanism of toxin disposition in renal disease state.
PMCID: PMC3046181  PMID: 21386972
11.  Plasmodium vivax Tryptophan-Rich Antigen PvTRAg33.5 Contains Alpha Helical Structure and Multidomain Architecture 
PLoS ONE  2011;6(1):e16294.
Tryptophan-rich proteins from several malarial parasites have been identified where they play an important role in host-parasite interaction. Structural characterization of these proteins is needed to develop them as therapeutic targets. Here, we describe a novel Plasmodium vivax tryptophan-rich protein named PvTRAg33.5. It is expressed by blood stage(s) of the parasite and its gene contains two exons. The exon 1 encodes for a 23 amino acids long putative signal peptide which is likely to be cleaved off whereas the exon 2 encodes for the mature protein of 252 amino acids. The mature protein contains B-cell epitopes which were recognized by the human immune system during P.vivax infection. The PvTRAg33.5 contains 24 (9.5%) tryptophan residues and six motifs whose patterns were similar among tryptophan-rich proteins. The modeled structure of the PvTRAg33.5 consists of a multidomain architecture which is stabilized by the presence of large number of tryptophan residues. The recombinant PvTRAg33.5 showed predominantly α helical structure and alpha helix to beta sheet transition at pH below 4.5. Protein acquires an irreversible non-native state at temperature more than 50°C at neutral pH. Its secondary and tertiary structures remain stable in the presence of 35% alcohol but these structures are destabilized at higher alcohol concentrations due to the disturbance of hydrophobic interactions between tryptophanyl residues. These structural changes in the protein might occur during its translocation to interact with other proteins at its final destination for biological function such as erythrocyte invasion.
PMCID: PMC3024423  PMID: 21283717
12.  Characterization of lectins and their specificity in carcinomas—An appraisal 
Lectins, a group of specific glycoproteins present in animal as well as plant cells, are used as differentiating markers to study cancers and metastatic cell lines. This property of lectins depends on the process of cellular glycosylation. Glycosylation of some of the extracellular membrane proteins and lipids maintains the cell/cell and cell/matrix interactions. Chemical alterations in glycosylation play an important role in the metastatic behavior of tumor cells. Carbohydrate residues of the membrane glycoproteins can be detected using lectins due to their binding specificity to carbohydrates. Lectins, therefore have gained an importance in the field of cancer research. Galectins, a specialized group of lectin like proteins that are Ca+ independent and galactoside binding, are also considered as differentiation markers in some specific cancers like the carcinomas of thyroid.
Thus the use of lectins and galectins to identify specific carbohydrates present on cell surface help in invasion and metastasis processes.
PMCID: PMC3453863  PMID: 23105409
lectins; metastasis; cellular glycosylation; prognostic markers; galectins

Results 1-12 (12)