Publications by authors named "Soumyananda Chakraborti"

31 Publications

A single residue can modulate nanocage assembly in salt dependent ferritin.

Nanoscale 2021 Jul;13(27):11932-11942

Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7A, 30-392 Krakow, Poland.

Cage forming proteins have numerous potential applications in biomedicine and biotechnology, where the iron storage ferritin is a widely used example. However, controlling ferritin cage assembly/disassembly remains challenging, typically requiring extreme conditions incompatible with many desirable cargoes, particularly for more fragile biopharmaceuticals. Recently, a ferritin from the hyperthermophile bacterium Thermotoga maritima (TmFtn) has been shown to have reversible assembly under mild conditions, offering greater potential biocompatibility in terms of cargo access and encapsulation. Like Archeoglobus fulgidus ferritin (AfFtn), TmFtn forms 24mer cages mediated by metal ions (Mg2+). We have solved the crystal structure of the wild type TmFtn and several mutants displaying different assembly/disassembly properties. These data combined with other biophysical studies allow us to suggest candidate interfacial amino acids crucial in controlling assembly. This work deepens our understanding of how these ferritin complexes assemble and is a useful step towards production of triggerable ferritins in which these properties can be finely designed and controlled.
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http://dx.doi.org/10.1039/d1nr01632fDOI Listing
July 2021

Multivalent Display of SARS-CoV-2 Spike (RBD Domain) of COVID-19 to Nanomaterial, Protein Ferritin Nanocages.

Biomolecules 2021 02 17;11(2). Epub 2021 Feb 17.

University of Gdansk, International Centre for Cancer Vaccine Science, ul. Kładki 24, 80-822 Gdansk, Poland.

SARS-CoV-2, or COVID-19, has a devastating effect on our society, both in terms of quality of life and death rates; hence, there is an urgent need for developing safe and effective therapeutics against SARS-CoV-2. The most promising strategy to fight against this deadly virus is to develop an effective vaccine. Internalization of SARS-CoV-2 into the human host cell mainly occurs through the binding of the coronavirus spike protein (a trimeric surface glycoprotein) to the human angiotensin-converting enzyme 2 (ACE2) receptor. The spike-ACE2 protein-protein interaction is mediated through the receptor-binding domain (RBD) of the spike protein. Mutations in the spike RBD can significantly alter interactions with the ACE2 host receptor. Due to its important role in virus transmission, the spike RBD is considered to be one of the key molecular targets for vaccine development. In this study, a spike RBD-based subunit vaccine was designed by utilizing a ferritin protein nanocage as a scaffold. Several fusion protein constructs were designed in silico by connecting the spike RBD via a synthetic linker (different sizes) to different ferritin subunits (H-ferritin and L-ferritin). The stability and the dynamics of the engineered nanocage constructs were tested by extensive molecular dynamics simulation (MDS). Based on our MDS analysis, a five amino acid-based short linker (S-Linker) was the most effective for displaying the spike RBD over the surface of ferritin. The behavior of the spike RBD binding regions from the designed chimeric nanocages with the ACE2 receptor was highlighted. These data propose an effective multivalent synthetic nanocage, which might form the basis for new vaccine therapeutics designed against viruses such as SARS-CoV-2.
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http://dx.doi.org/10.3390/biom11020297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923090PMC
February 2021

Electrostatic Self-Assembly of Protein Cage Arrays.

Methods Mol Biol 2021 ;2208:123-133

Biohybrid Materials, Department of Bioproducts and Biosystems, Aalto University, Aalto, Finland.

Protein and peptide cages are nanoscale containers, which are of particular interest in nanoscience due to their well-defined dimensions and enclosed central cavities that can be filled with material that is protected from the outside environment. Ferritin is a typical example of protein cage, formed by 24 polypeptide chains that self-assemble into a hollow, roughly spherical protein cage with external and internal diameters of approximately 12 nm and 8 nm, respectively. The interior cavity of ferritin provides a unique reaction vessel to carry out reactions separated from the exterior environment. In nature, the cavity is utilized for sequestration and biomineralization to render iron inert and safe by shielding from the external environment. Materials scientists have been inspired by this system and exploited a range of ferritin superfamily proteins as supramolecular templates to encapsulate cargoes ranging from cancer drugs to therapeutic proteins. Interesting possibilities arise if such containers can themselves be arranged into even higher-order structures such as crystalline arrays. Here, we describe how crystalline arrays of negatively charged ferritin protein cages can be built by taking advantage of electrostatic interactions with cationic gold nanoparticles.
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http://dx.doi.org/10.1007/978-1-0716-0928-6_8DOI Listing
March 2021

A bacteriophage mimic of the bacterial nucleoid-associated protein Fis.

Biochem J 2020 04;477(7):1345-1362

Department of Biosciences, Durham University, Stockton Road, Durham DH1 3LE, U.K.

We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein (NAP) involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15 bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported the identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like NAP that likely influences phage excision-integration reactions or bacterial gene expression.
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http://dx.doi.org/10.1042/BCJ20200146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7166090PMC
April 2020

Self-Assembly of Ferritin: Structure, Biological Function and Potential Applications in Nanotechnology.

Adv Exp Med Biol 2019 ;1174:313-329

Department of Biochemistry, Bose Institute, Kolkata, India.

Protein cages are normally formed by the self-assembly of multiple protein subunits and ferritin is a typical example of a protein cage structure. Ferritin is a ubiquitous multi-subunit iron storage protein formed by 24 polypeptide chains that self-assemble into a hollow, roughly spherical protein cage. Ferritin has external and internal diameters of approximately 12 nm and 8 nm, respectively. Functionally, ferritin performs iron sequestration and is highly conserved in evolution. The interior cavity of ferritin provides a unique reaction vessel to carry out reactions separated from the exterior environment. In nature, the cavity is utilized for sequestration of iron and bio-mineralization as a mechanism to render iron inert and safe from the external environment. Material scientists have been inspired by this system and exploited a range of ferritin superfamily proteins as supramolecular templates to encapsulate different carrier molecules ranging from cancer drugs to therapeutic proteins, in addition to using ferritin proteins as well-defined building blocks for fabrication. Besides the interior cavity, the exterior surface and sub-unit interface of ferritin can be modified without affecting ferritin assembly.
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http://dx.doi.org/10.1007/978-981-13-9791-2_10DOI Listing
November 2019

Three-Dimensional Protein Cage Array Capable of Active Enzyme Capture and Artificial Chaperone Activity.

Nano Lett 2019 06 30;19(6):3918-3924. Epub 2019 May 30.

Bionanoscience and Biochemistry Laboratory, Malopolska Centre of Biotechnology , Jagiellonian University , Gronostajowa 7A , 30-387 Krakow , Poland.

Development of protein cages for encapsulation of active enzyme cargoes and their subsequent arrangement into a controllable three-dimensional array is highly desirable. However, cargo capture is typically challenging because of difficulties in achieving reversible assembly/disassembly of protein cages in mild conditions. Herein we show that by using an unusual ferritin cage protein that undergoes triggerable assembly under mild conditions, we can achieve reversible filling with protein cargoes including an active enzyme. We demonstrate that these filled cages can be arrayed in three-dimensional crystal lattices and have an additional chaperone-like effect, increasing both thermostability and enzymatic activity of the encapsulated enzyme.
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http://dx.doi.org/10.1021/acs.nanolett.9b01148DOI Listing
June 2019

An ultra-stable gold-coordinated protein cage displaying reversible assembly.

Nature 2019 05 8;569(7756):438-442. Epub 2019 May 8.

Heddle Initiative Research Unit, RIKEN, Saitama, Japan.

Symmetrical protein cages have evolved to fulfil diverse roles in nature, including compartmentalization and cargo delivery, and have inspired synthetic biologists to create novel protein assemblies via the precise manipulation of protein-protein interfaces. Despite the impressive array of protein cages produced in the laboratory, the design of inducible assemblies remains challenging. Here we demonstrate an ultra-stable artificial protein cage, the assembly and disassembly of which can be controlled by metal coordination at the protein-protein interfaces. The addition of a gold (I)-triphenylphosphine compound to a cysteine-substituted, 11-mer protein ring triggers supramolecular self-assembly, which generates monodisperse cage structures with masses greater than 2 MDa. The geometry of these structures is based on the Archimedean snub cube and is, to our knowledge, unprecedented. Cryo-electron microscopy confirms that the assemblies are held together by 120 S-Au-S staples between the protein oligomers, and exist in two chiral forms. The cage shows extreme chemical and thermal stability, yet it readily disassembles upon exposure to reducing agents. As well as gold, mercury(II) is also found to enable formation of the protein cage. This work establishes an approach for linking protein components into robust, higher-order structures, and expands the design space available for supramolecular assemblies to include previously unexplored geometries.
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http://dx.doi.org/10.1038/s41586-019-1185-4DOI Listing
May 2019

Molecular features of interaction involving hen egg white lysozyme immobilized on graphene oxide and the effect on activity.

Int J Biol Macromol 2018 Dec 13;120(Pt B):2390-2398. Epub 2018 Sep 13.

Department of Biochemistry, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054, India; Bioinformatics Centre, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054, India. Electronic address:

Nanomaterials, such as graphene oxide (GO) are being studied to decipher their suitability in biomedical applications. This study investigate the effect on structure and function of hen egg white lysozyme (HEWL) adsorbed on GO, using various biophysical techniques. In spite of there being not much change in the structure, the catalytic activity is reduced significantly. Fluorescence quenching indicates complex formation. Fluorescence lifetime measurement suggests that GO binds at or near the active site close to Trp62 and Trp108. Heat change associated with HEWL-GO interaction suggests hydrogen bond along with van der Waals and electrostatic interactions are involved in the HEWL-GO complex. Molecular docking indicates binding of GO at the active site corroborating experimental findings. Molecular dynamics simulations indicate that the blocking of the active site affects the flexibility of the surrounding residues and contribute to the reduction of the activity. Unfolding experiments indicate that HEWL is more prone to thermal instability in presence of GO. Together, the results obtained established molecular details of HEWL-GO interaction and might be useful in eventual biomedical applications of GO.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.09.007DOI Listing
December 2018

Effect of ZnO quantum dots on Escherichia coli global transcription regulator: A molecular investigation.

Int J Biol Macromol 2018 Oct 2;117:1280-1288. Epub 2018 Jun 2.

Department of Biochemistry, Bose Institute, P-1/12, C.I.T. Scheme VIIM, Kolkata 700054, India; Bionanoscience and Biochemistry Laboratory, Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland. Electronic address:

ZnO quantum dots (QDs) are very well known for their antimicrobial activity against several bacteria, however, we still do not know any protein targets of ZnO QDs. In order to determine possible protein target, interaction of ZnO QDs was studied with CRP (Cyclic AMP Receptor Protein), a global transcription regulator protein. Binding between ZnO QDs and E. coli CRP was mainly studied by isothermal titration calorimetry (ITC), structural changes of protein were monitored by fluorescence and circular dichroism spectroscopy, and in-vitro transcription assay was used to asses CRP activity. Result shows that both electrostatic and hydrophobic interactions are involved in CRP-ZnO binding. Different spectroscopic investigation revealed that ZnO binding to CRP leads to extensive unfolding and destabilization, which ultimately leads to protein aggregation. It was also observed that in presence of ZnO dimerization ability of CRP was sharply reduced. In-vitro transcription assay also shows that CRP activity gets severely compromised on ZnO binding. All our data suggests that ZnO QD binding to CRP and consequent structural and functional changes most probably plays a crucial role in ZnO QD induced antimicrobial action.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.06.001DOI Listing
October 2018

Natural and artificial protein cages: design, structure and therapeutic applications.

Curr Opin Struct Biol 2017 04 27;43:148-155. Epub 2017 Mar 27.

Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Japan. Electronic address:

Advanced electron microscopy techniques have been used to solve many viral capsid structures. The resulting detailed structural knowledge contributes to understanding of the mechanisms of self-assembly, maturation pathways and virion-host cell interactions. It also acts as inspiration for design and production of capsid-like artificial protein cages. Both natural and artificial cages have potential uses in medicine including as vaccines and in drug delivery. For vaccines, virus-like particles formed only from outer virion shells, lacking genetic material, offer the simplest basis for development, while encapsulation of target molecules inside protein cages is potentially more challenging. Here we review advances in cryo-electron microscopy with particular reference to viral capsid structures. We then consider why knowledge of these structures is useful, giving examples of their utilization as encapsulation and vaccine agents. Finally we look at the importance of structural techniques including cryo-EM in the rapidly progressing field of designed protein cages.
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http://dx.doi.org/10.1016/j.sbi.2017.03.007DOI Listing
April 2017

PEG-functionalized zinc oxide nanoparticles induce apoptosis in breast cancer cells through reactive oxygen species-dependent impairment of DNA damage repair enzyme NEIL2.

Free Radic Biol Med 2017 02 7;103:35-47. Epub 2016 Dec 7.

Department of Biochemistry, Bose Institute, P-1/12 C.I.T. Scheme, VIIM, Kolkata 700054, India. Electronic address:

We find that PEG functionalized ZnO nanoparticles (NP) have anticancer properties primarily because of ROS generation. Detailed investigation revealed two consequences depending on the level of ROS - either DNA damage repair or apoptosis - in a time-dependent manner. At early hours of treatment, NP promote NEIL2-mediated DNA repair process to counteract low ROS-induced DNA damage. However, at late hours these NP produce high level of ROS that inhibits DNA repair process, thereby directing the cell towards apoptosis. Mechanistically at low ROS conditions, transcription factor Sp1 binds to the NEIL2 promoter and facilitates its transcription for triggering a 'fight-back mechanism' thereby resisting cancer cell apoptosis. In contrast, as ROS increase during later hours, Sp1 undergoes oxidative degradation that decreases its availability for binding to the promoter thereby down-regulating NEIL2 and impairing the repair mechanism. Under such conditions, the cells strategically switch to the p53-dependent apoptosis.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.11.048DOI Listing
February 2017

The emerging role of the tubulin code: From the tubulin molecule to neuronal function and disease.

Cytoskeleton (Hoboken) 2016 Oct 9;73(10):521-550. Epub 2016 May 9.

Children's National Health System, Center for Neuroscience Research, Washington, District of Columbia.

Across different cell types and tissues, microtubules are assembled from highly conserved dimers of α- and β-tubulin. Despite their highly similar structures, microtubules have functional heterogeneity, generated either by the expression of different tubulin genes, encoding distinct isotypes, or by posttranslational modifications of tubulin. This genetically encoded and posttranslational generated heterogeneity of tubulin-the "tubulin code"-has the potential to modulate microtubule structure, dynamics, and interactions with associated proteins. The tubulin code is therefore believed to regulate microtubule functions on a cellular and sub-cellular level. This review highlights the importance of the tubulin code for tubulin structure, as well as on microtubule dynamics and functions in neurons. It further summarizes recent developments in the understanding of mutations in tubulin genes, and how they are linked to neurodegenerative and neurodevelopmental disorders. The current advances in the knowledge of the tubulin code on the molecular and the functional level will certainly lead to a better understanding of how complex signaling events control microtubule functions, especially in cells of the nervous system. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/cm.21290DOI Listing
October 2016

The antimicrobial activity of ZnO nanoparticles against Vibrio cholerae: Variation in response depends on biotype.

Nanomedicine 2016 08 10;12(6):1499-509. Epub 2016 Mar 10.

Department of Biochemistry, Bose Institute, P1/12 CIT Scheme VIIM, Kolkata, India. Electronic address:

The potency of zinc oxide nanoparticles (NPs), with a core size of ~7-10nm, to inhibit cholera disease was investigated by demonstrating the effect on two biotypes (classical and El Tor) of O1 serogroup of Vibrio cholerae-El Tor was more susceptible both in planktonic and in biofilm forms. Interaction with ZnO NP results in deformed cellular architecture. Increased fluidity and depolarization of membrane, and protein leakage further confirmed the damages inflicted on Vibrio by NP. NP was shown to produce reactive oxygen species (ROS) and induce DNA damage. These results suggest that the antibacterial mechanism of ZnO action is most likely due to generation of ROS and disruption of bacterial membrane. The antimicrobial efficacy of NP has been validated in animal model. The synergistic action of NP and antibiotic suggests an alternative for the treatment of cholera.
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http://dx.doi.org/10.1016/j.nano.2016.02.006DOI Listing
August 2016

Antibacterial and DNA degradation potential of silver nanoparticles synthesized via green route.

Int J Biol Macromol 2015 Sep 16;80:455-9. Epub 2015 Jul 16.

Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721102, West Bengal, India. Electronic address:

Silver nanoparticles (AgNPs) were synthesized using a hetero polysaccharide (PS) isolated from Lentinus squarrosulus (Mont.) Singer. The polysaccharide fraction (consisting of glucose, fucose and galactose) serves the role of both reducing as well as stabilizing agent. UV-vis spectroscopy showed maximum absorbance at 407 nm due to surface plasmon resonance. High resolution transmission electron microscopy (HRTEM) exhibited that the average diameter of the nanoparticles was 2.78±1.47 nm. The XRD analysis revealed face-centered cubic (fcc) geometry of silver nanoparticles. Antibacterial activity of the AgNPs-PS conjugate was tested against multiple antibiotics resistant (MAR) Escherichia coli strain MREC33 and found that the killing was due to generation of reactive oxygen species (ROS). Internalization of AgNPs-PS conjugate along with its DNA degradation capability was demonstrated using flow cytometry. AgNPs-PS conjugates showed negligible toxicity to human RBCs. This LD50 dosage of AgNPs-PS conjugates in combination with each of the four antibiotics (ampicillin, azithromycin, kanamycin and netilmicin) to which E. coli MREC33 was resistant, showed synergistic effect to inhibit complete bacterial growth.
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http://dx.doi.org/10.1016/j.ijbiomac.2015.07.028DOI Listing
September 2015

Flexibility in the N-terminal actin-binding domain: clues from in silico mutations and molecular dynamics.

Proteins 2015 Apr 5;83(4):696-710. Epub 2015 Feb 5.

Department of Biochemistry, Bose Institute, Kolkata, West Bengal, 700054, India.

Dystrophin is a long, rod-shaped cytoskeleton protein implicated in muscular dystrophy (MDys). Utrophin is the closest autosomal homolog of dystrophin. Both proteins have N-terminal actin-binding domain (N-ABD), a central rod domain and C-terminal region. N-ABD, composed of two calponin homology (CH) subdomains joined by a helical linker, harbors a few disease causing missense mutations. Although the two proteins share considerable homology (>72%) in N-ABD, recent structural and biochemical studies have shown that there are significant differences (including stability, mode of actin-binding) and their functions are not completely interchangeable. In this investigation, we have used extensive molecular dynamics simulations to understand the differences and the similarities of these two proteins, along with another actin-binding protein, fimbrin. In silico mutations were performed to identify two key residues that might be responsible for the dynamical difference between the molecules. Simulation points to the inherent flexibility of the linker region, which adapts different conformations in the wild type dystrophin. Mutations T220V and G130D in dystrophin constrain the flexibility of the central helical region, while in the two known disease-causing mutants, K18N and L54R, the helicity of the region is compromised. Phylogenetic tree and sequence analysis revealed that dystrophin and utrophin genes have probably originated from the same ancestor. The investigation would provide insight into the functional diversity of two closely related proteins and fimbrin, and contribute to our understanding of the mechanism of MDys.
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http://dx.doi.org/10.1002/prot.24767DOI Listing
April 2015

Microtubule +TIP protein EB1 binds to GTP and undergoes dissociation from dimer to monomer on binding GTP.

Biochemistry 2014 Sep 21;53(34):5551-7. Epub 2014 Aug 21.

School of Biology, Indian Institute of Science Education and Research, Thiruvananthapuram , CET Campus, Thiruvananthapuram, Kerala 695016, India.

The +TIP protein EB1 autonomously tracks the growing plus end of microtubules and regulates plus-end dynamics. Previous studies have indicated that EB1 can recognize GTP-bound tubulin structures at the plus end, and it localizes on the microtubule surface at a site close to the exchangeable GTP-binding site of tubulin. Although the GTP-dependent structural change in tubulin has been demonstrated to be a critical determinant for recognition of plus ends by EB1, the effect of GTP on the structure of EB1 has remained unclear. Here, we have used spectroscopic, calorimetric, and biochemical methods to analyze the effect of GTP on EB1 in vitro. Isothermal titration calorimetry and tryptophan fluorescence quenching experiments demonstrated that EB1 binds to GTP with a dissociation constant ~30 μM. Circular dichroism measurements showed that EB1 undergoes changes in its secondary structure on binding GTP. Size-exclusion chromatography and urea-induced unfolding analyses revealed that GTP binding induces dissociation of the EB1 dimer to monomers. Size-exclusion chromatography followed by biochemical analysis further determined that EB1-GTP binding involves association of approximately one molecule of GTP per EB1 monomer. The results reveal a hitherto unknown GTP-dependent mechanism of dimer-to-monomer transition in EB1 and further implicate its possible role in regulating the stability of the EB1 dimer vs monomer as well as plus-end regulation in cells.
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http://dx.doi.org/10.1021/bi5007942DOI Listing
September 2014

Bactericidal effect of polyethyleneimine capped ZnO nanoparticles on multiple antibiotic resistant bacteria harboring genes of high-pathogenicity island.

Colloids Surf B Biointerfaces 2014 Sep 22;121:44-53. Epub 2014 May 22.

Department of Biochemistry, Bose Institute, P1/12 CIT Scheme VIIM, Kolkata 700054, India. Electronic address:

Zinc oxide nanoparticles (ZnO-NP) were synthesized by alcoholic route using zinc acetate as the precursor material and lithium hydroxide as hydrolyzing agent. Further ZnO-PEI NP (derivative of ZnO-NP) was made in aqueous medium using the capping agent polyethyleneimine (PEI). The nanoparticles were characterized by XRD measurements, TEM and other techniques; the weight % of coating shell in the polymer-capped particles was determined by TGA. ZnO-PEI NP is more soluble in water than the uncapped ZnO-NP, and forms a colloidal suspension in water. PEI-capped ZnO-NP exhibited better antibacterial activity when compared with that of uncapped ZnO-NP against a range of multiple-antibiotic-resistant (MAR) Gram-negative bacterial strains harboring genes of high-pathogenicity island. ZnO-NP effectively killed these microorganisms by generating reactive oxygen species (ROS) and damaging bacterial membrane. ZnO-PEI NP at LD50 dose in combination with tetracycline showed synergistic effect to inhibit tetracycline-resistant Escherichia coli MREC33 growth by 80%. These results open up a new vista in therapeutics to use antibiotics (which have otherwise been rendered useless against MAR bacteria) in combination with minimized dosage of nanoparticles for the more effective control of MAR pathogenic bacteria.
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http://dx.doi.org/10.1016/j.colsurfb.2014.03.044DOI Listing
September 2014

Green synthesis of silver nanoparticles using glucan from mushroom and study of antibacterial activity.

Int J Biol Macromol 2013 Nov 26;62:439-49. Epub 2013 Sep 26.

Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721102, West Bengal, India.

This work demonstrates synthesis of silver nanoparticles (AgNPs) using glucan isolated from a mushroom Pleurotus florida blue variant. UV-vis spectroscopy showed maximum absorbance at 425 nm due to surface plasmon resonance of AgNPs. Average diameter of the synthesized AgNPs was 2.445 ± 1.08 nm as revealed from TEM analysis. XRD analysis confirmed the face-centered cubic (fcc) crystalline structure of metallic silver. The synthesized AgNPs-glucan conjugates exhibited antibacterial activity against multiple antibiotic resistant (MAR) bacterium Klebsiella pneumoniae YSI6A and the activity was possibly due to damage of cellular macromolecules by the generation of reactive oxygen species (ROS) which was supported by observed degradation of bacterial DNA. Decrease of bactericidal effect of AgNPs-glucan conjugates in dose-dependent manner in presence of a ROS scavenger histidine further ascertained the involvement of ROS in antibacterial activity. AgNPs-glucan conjugates at LD50 dose caused least damage (0.68% hemolysis) to human RBCs. This particular dose of AgNPs-glucan conjugates in combination with each of the four antibiotics (ampicillin, azithromycin, cefepime and tetracycline) to which K. pneumoniae YSI6A was resistant, showed synergistic effect to inhibit almost 100% bacterial growth. It thus opens an avenue to use antibiotics in combination with minimum dosages of AgNPs-glucan conjugates to control MAR bacteria.
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http://dx.doi.org/10.1016/j.ijbiomac.2013.09.019DOI Listing
November 2013

Stable and potent analogues derived from the modification of the dicarbonyl moiety of curcumin.

Biochemistry 2013 Oct 10;52(42):7449-60. Epub 2013 Oct 10.

Department of Biochemistry, Bose Institute , P-1/12 CIT Scheme VIIM, Kolkata 700054, India.

Curcumin has shown promising therapeutic utilities for many diseases, including cancer; however, its clinical application is severely limited because of its poor stability under physiological conditions. Here we find that curcumin also loses its activity instantaneously in a reducing environment. Curcumin can exist in solution as a tautomeric mixture of keto and enol forms, and the enol form was found to be responsible for the rapid degradation of the compound. To increase the stability of curcumin, several analogues were synthesized in which the diketone moiety of curcumin was replaced by isoxazole (compound 2) and pyrazole (compound 3) groups. Isoxazole and pyrazole curcumins were found to be extremely stable at physiological pH, in addition to reducing atmosphere, and they can kill cancer cells under serum-depleted condition. Using molecular modeling, we found that both compounds 2 and 3 could dock to the same site of tubulin as the parent molecule, curcumin. Interestingly, compounds 2 and 3 also show better free radical scavenging activity than curcumin. Altogether, these results strongly suggest that compounds 2 and 3 could be good replacements for curcumin in future drug development.
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http://dx.doi.org/10.1021/bi400734eDOI Listing
October 2013

The effect of the binding of ZnO nanoparticle on the structure and stability of α-lactalbumin: a comparative study.

J Phys Chem B 2013 Oct 22;117(43):13397-408. Epub 2013 Oct 22.

Department of Biochemistry, Bose Institute , Kolkata 700054, India.

Nanoparticles (NPs), when exposed to biofluids, become coated with proteins. As the protein is adsorbed on the surface, the extent of adsorption and the consequent effect on protein conformation and activity depend on the chemical nature, shape, and size of the nanoparticle. We have carried out a detailed study on the interaction of α-lactalbumin (a protein which forms the regulatory subunit of lactose synthase) with zinc oxide nanoparticles. The NPs were prepared by the sol-gel route and characterized by transmission electron microscopy, X-ray diffraction, UV-visible, and photoluminescence spectroscopy. ZnO particles were found to have a size of 4-7 nm with hexagonal structure. The interaction of protein with NP was examined using a combination of spectroscopic and computational methods. The binding was studied by ITC (isothermal calorimetry), and the result revealed that the complexation is mostly entropy driven and involves hydrophobic interaction. There is alteration in secondary structures in protein on binding ZnO nanoparticle, as revealed by circular dichroism (CD) and Fourier transform infrared spectroscopy (FITR). Finally, a comparison of structure, function, and stability of the α-lactalbumin-NP complex has been made by binding ZnO to other model proteins to get a better insight into the process of protein nanoparticle interaction. The present study thus provides useful insights into issues such as protein-nanoparticle recognition.
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http://dx.doi.org/10.1021/jp404411bDOI Listing
October 2013

The molecular basis of inactivation of metronidazole-resistant Helicobacter pylori using polyethyleneimine functionalized zinc oxide nanoparticles.

PLoS One 2013 8;8(8):e70776. Epub 2013 Aug 8.

Department of Biochemistry, Bose Institute, Kolkata, India.

In view of the world wide prevalence of Helicobacter pylori infection, its potentially serious consequences, and the increasing emergence of antibiotic resistant H. pylori strains there is an urgent need for the development of alternative strategies to combat the infection. In this study it has been demonstrated that polyethyleneimine (PEI) functionalized zinc oxide (ZnO) nanoparticles (NPs) inhibit the growth of a metronidazole-resistant strain of H. pylori and the molecular basis of the anti-bacterial activity of ZnO-PEI NP has been investigated. The ZnO-PEI NP was synthesized using a wet chemical method with a core size of approximately 3-7 nm. Internalization and distribution of ZnO-PEI NP without agglomeration was observed in H. pylori cytosol by electron microscopy. Several lines of evidence including scanning electron microscopy, propidium iodide uptake and ATP assay indicate severe membrane damage in ZnO-PEI NP treated H. pylori. Intracellular ROS generation increased rapidly following the treatment of H. pylori with ZnO-PEI NP and extensive degradation of 16S and 23S rRNA was observed by quantitative reverse-transcriptase PCR. Finally, considerable synergy between ZnO-PEI NP and antibiotics was observed and it has been demonstrated that the concentration of ZnO-PEI NP (20 µg/ml) that is non-toxic to human cells could be used in combination with sub-inhibitory concentrations of antibiotics for the inhibition of H. pylori growth.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070776PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3738536PMC
April 2014

Flocculating property of extracellular polymeric substances produced by a biofilm-forming bacterium Acinetobacter junii BB1A.

Appl Biochem Biotechnol 2012 Nov 12;168(6):1621-34. Epub 2012 Sep 12.

Omics Laboratory, Department of Biotechnology, University of North Bengal, Siliguri, West Bengal 734013, India.

Extracellular polymeric substances (EPS) produced by a biofilm-producing bacterium Acinetobacter junii BB1A were characterized. Purified EPS on analysis revealed neutral sugar (73.21 %), uronic acid (10.12 %), amino sugars (0.23 %), α- amino acids (11.13 %), and aromatic amino acids (1.23 %). Infrared spectrometry revealed the presence of hydroxyl, carboxyl, and amide groups. The average molecular weight of the polysaccharide (PS) fraction of EPS was ~2×10(5). Gas liquid chromatography analysis of PS revealed the presence of three main sugar residues, namely, mannose, galactose, and arabinose (molar ratio of 3:1:1). Cation-independent flocculation above 90 % was observed in the pH range of 4-5 with EPS dosage of 30 mgl(-1) at 20 °C. The emulsifying activity of EPS was 66.6 % with toluene, 60 % with n-hexadecane, 53.3 % with olive oil, and least activity of 13.3 % with kerosene. Proteinase K- and trichloroacetic acid-treated EPS showed reduction in flocculation and emulsification, suggesting the significant role of protein component. Energy dispersive X-ray spectroscopy was used to provide direct evidence of biosorption of Co(II), Cd(II), and Hg(II) by EPS.
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http://dx.doi.org/10.1007/s12010-012-9883-5DOI Listing
November 2012

Partial characterization and flocculating behavior of an exopolysaccharide produced in nutrient-poor medium by a facultative oligotroph Klebsiella sp. PB12.

J Biosci Bioeng 2013 Jan 1;115(1):76-81. Epub 2012 Sep 1.

Department of Biotechnology, Omics Laboratory, University of North Bengal, Siliguri 734013, West Bengal, India.

A facultative oligotrophic strain from the water sample of River Mahananda, Siliguri India was selected for its property to produce exopolysaccharide (EPS) in nutrient-poor (oligotrophic) medium. Viability assay of the strain was performed in sterile liquid LB, R2A, river water and diluted (10(-3)) LB at 30°C and pH 7 to understand oligotrophy. The selected strain was identified by 16S rRNA gene sequencing and designated as Klebsiella sp. PB12. Phylogenetic analysis showed its closest relationship with Klebsiella variicola ATCC BAA-830(T). Purification of EPS was performed by ethanol precipitation, dialysis and freeze-drying. Chemical analysis revealed that purified EPS was mainly composed of 72.32% (w/w) neutral sugar and 14.12% (w/w) uronic acids. Fourier transform infrared (FT-IR) spectroscopy indicated the presence of hydroxyl, carboxylic and methoxyl functional groups. The optimal dosages for flocculation of activated carbon suspension were 17 mg/l EPS and 4 mM CaCl(2). EPS showed flocculating rate of above 80% over a wide range of pH (pH 3-10) whereas, more than 90% rate was noted in the temperature range (10-50°C) tested in presence of CaCl(2). Moreover, EPS showed characteristic emulsifying activity with toluene (66.6%), n-hexadecane (65%), olive oil (63.3%) and kerosene (50%). The apparent molecular weight of the EPS was ~2 × 10(5) Da.
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http://dx.doi.org/10.1016/j.jbiosc.2012.08.006DOI Listing
January 2013

Discrimination of ligands with different flexibilities resulting from the plasticity of the binding site in tubulin.

Biochemistry 2012 Sep 27;51(36):7138-48. Epub 2012 Aug 27.

Department of Biochemistry, Bose Institute, Kolkata 700054, India.

Tubulin, an α,β heterodimer, has four distinct ligand binding sites (for paclitaxel, peloruside/laulimalide, vinca, and colchicine). The site where colchicine binds is a promising drug target for arresting cell division and has been observed to accommodate compounds that are structurally diverse but possess comparable affinity. This investigation, using two such structurally different ligands as probes (one being colchicine itself and another, TN16), aims to provide insight into the origin of this diverse acceptability to provide a better perspective for the design of novel therapeutic molecules. Thermodynamic measurements reveal interesting interplay between entropy and enthalpy. Although both these parameters are favourable for TN16 binding (ΔH < 0, ΔS > 0), but the magnitude of entropy has the determining role for colchicine binding as its enthalpic component is destabilizing (ΔH > 0, ΔS > 0). Molecular dynamics simulation provides atomistic insight into the mechanism, pointing to the inherent flexibility of the binding pocket that can drastically change its shape depending on the ligand that it accepts. Simulation shows that in the complexed states both the ligands have freedom to move within the binding pocket; colchicine can switch its interactions like a "flying trapeze", whereas TN16 rocks like a "swing cradle", both benefiting entropically, although in two different ways. Additionally, the experimental results with respect to the role of solvation entropy correlate well with the computed difference in the hydration: water molecules associated with the ligands are released upon complexation. The complementary role of van der Waals packing versus flexibility controls the entropy-enthalpy modulations. This analysis provides lessons for the design of new ligands that should balance between the "better fit" and "flexibility"', instead of focusing only on the receptor-ligand interactions.
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http://dx.doi.org/10.1021/bi300474qDOI Listing
September 2012

Interaction of polyethyleneimine-functionalized ZnO nanoparticles with bovine serum albumin.

Langmuir 2012 Jul 19;28(30):11142-52. Epub 2012 Jul 19.

Department of Biochemistry, Bose Institute, Kolkata, India.

In biological fluids, nanoparticles are always surrounded by proteins. As the protein is adsorbed on the surface, the extent of adsorption and the effect on the protein conformation and stability are dependent on the chemical nature, shape, and size of the nanoparticle (NP). We have carried out a detailed investigation on the interaction of bovine serum albumin (BSA) with polyethyleneimine-functionalized ZnO nanoparticles (ZnO-PEI). ZnO-PEI was synthesized using a wet chemical method with a core size of ~3-7 nm (from transmission electron microscopy). The interaction of BSA with ZnO-PEI was examined using a combination of calorimetric, spectroscopic, and computational techniques. The binding was studied by ITC (isothermal titration calorimetry), and the result revealed that the complexation is enthalpy-driven, indicating the possible involvement of electrostatic interaction. To investigate the nature of the interaction and the location of the binding site, a detailed domain-wise surface electrostatic potential calculation was performed using adaptive Poisson-Boltzmann software (APBS). The result shows that the protein surface can bind the nanoparticle. On binding ZnO-PEI, the protein gets destabilized to some extent, as displayed by CD (circular dichroism) and FTIR (Fourier transform infrared) spectroscopy. Chemical and thermal denaturation of BSA, when carried out in the presence of ZnO-PEI, also indicated a small perturbation in the protein structure. A comparison of the enthalpy and entropy components of binding with those derived for the interaction of BSA with ZnO nanoparticles explains the effect of hydrophilic cationic species attached on the NP surface. The effect of the NP surface modification on the structure and stability of BSA would find useful applications in nanobiotechnology.
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http://dx.doi.org/10.1021/la3007603DOI Listing
July 2012

CIL-102 binds to tubulin at colchicine binding site and triggers apoptosis in MCF-7 cells by inducing monopolar and multinucleated cells.

Biochem Pharmacol 2012 Sep 15;84(5):633-45. Epub 2012 Jun 15.

School of Biology, Indian Institute of Science Education and Research, Thiruvananthapuram, CET Campus, Thiruvananthapuram 695016, Kerala, India.

A plant dictamine analog, 1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone (CIL-102) has been shown to exert potent anti-tumor activity. In this study, we examined the mode of interaction of CIL-102 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. CIL-102 bound to tubulin at a single site with a dissociation constant ~0.4 μM. Isothermal titration calorimetry revealed that CIL-102-tubulin interaction is highly enthalpy driven and that the binding affords a large negative heat capacity change (ΔC(p) = -790 cal mol(-1) K(-1)) with an enthalpy-entropy compensation. An analysis of the modified Dixon plot suggested that CIL-102 competitively inhibited the binding of podophyllotoxin, a colchicine-binding site agent, to tubulin. Computational modeling indicated that CIL-102 binds exclusively at the β-subunit of tubulin and that CIL-102 and colchicine partially share their binding sites on tubulin. It bound to tubulin reversibly and the binding was estimated to be ~1000 times faster than that of colchicine. CIL-102 potently inhibited the proliferation of MCF-7 cells, induced monopolar spindle formation and multi-nucleation. At half-maximal inhibitory concentration, the spindle microtubules were visibly depolymerized and disorganized. CIL-102 reduced the inter-polar distances of bipolar mitotic cells indicating that it impaired microtubule-kinetochore attachments. CIL-102-treatment induced apoptosis in MCF-7 cells in association with increased nuclear accumulation of p53 and p21 suggesting that apoptosis is triggered through a p53-p21 dependent pathway. The results indicated that CIL-102 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided important insights into the differential mode of tubulin binding by CIL-102 and colchicine.
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http://dx.doi.org/10.1016/j.bcp.2012.06.008DOI Listing
September 2012

The anticancer activity of chloroquine-gold nanoparticles against MCF-7 breast cancer cells.

Colloids Surf B Biointerfaces 2012 Jun 6;95:195-200. Epub 2012 Mar 6.

National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi 110012, India.

In the present study, 11-mercaptoundecanoic acid-modified gold nanoparticles (∼7 nm) were conjugated with chloroquine to explore their potential application in cancer therapeutics. The anticancer activity of chloroquine-gold nanoparticle conjugates (GNP-Chl) was demonstrated in MCF-7 breast cancer cells. The MCF-7 cells were treated with different concentrations of GNP-Chl conjugates, and the cell viability was assayed using trypan blue, resulting in an IC(50) value of 30 ± 5 μg/mL. Flow cytometry analysis revealed that the major pathway of cell death was necrosis, which was mediated by autophagy. The drug release kinetics of GNP-Chl conjugates revealed the release of chloroquine at an acidic pH, which was quantitatively estimated using optical absorbance spectroscopy. The nature of stimuli-responsive drug release and the inhibition of cancer cell growth by GNP-Chl conjugates could pave the way for the design of combinatorial therapeutic agents, particularly nanomedicine, for the treatment of cancer.
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http://dx.doi.org/10.1016/j.colsurfb.2012.02.039DOI Listing
June 2012

Curcumin recognizes a unique binding site of tubulin.

J Med Chem 2011 Sep 29;54(18):6183-96. Epub 2011 Aug 29.

Department of Biochemistry, Bose Institute, Centenary Campus, P-1/12 CIT Scheme VIIM, Kolkata 700054, India.

Although curcumin is known for its anticarcinogenic properties, the exact mechanism of its action or the identity of the target receptor is not completely understood. Studies on a series of curcumin analogues, synthesized to investigate their tubulin binding affinities and tubulin self-assembly inhibition, showed that: (i) curcumin acts as a bifunctional ligand, (ii) analogues with substitution at the diketone and acetylation of the terminal phenolic groups of curcumin are less effective, (iii) a benzylidiene derivative, compound 7, is more effective than curcumin in inhibiting tubulin self-assembly. Cell-based studies also showed compound 7 to be more effective than curcumin. Using fluorescence spectroscopy we show that curcumin binds tubulin 32 Å away from the colchicine-binding site. Docking studies also suggests that the curcumin-binding site to be close to the vinblastine-binding site. Structure-activity studies suggest that the tridented nature of compound 7 is responsible for its higher affinity for tubulin compared to curcumin.
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http://dx.doi.org/10.1021/jm2004046DOI Listing
September 2011

The effect of zinc oxide nanoparticles on the structure of the periplasmic domain of the Vibrio cholerae ToxR protein.

FEBS J 2010 Oct 6;277(20):4184-94. Epub 2010 Sep 6.

Department of Biochemistry, Bose Institute, Kolkata, India.

Proteins adsorbed on nanoparticles (NPs) are being used as biosensors and in drug delivery. However, our understanding of the effect of NPs on the structure of proteins is still in a nascent state. In this work we report the unfolding behavior of the periplasmic domain of the ToxR protein (ToxRp) of Vibrio cholerae on zinc oxide (ZnO) nanoparticles with a diameter of 2.5 nm. This protein plays a crucial role in regulating the expression of several virulence factors in the pathogenesis of cholera. Thermodynamic analysis of the equilibrium of unfolding, induced both by urea and by guanidine hydrochloride (GdnHCl), and measured by fluorescence spectroscopy, revealed a two-state process. NPs increased the susceptibility of the protein to denaturation. The midpoints of transitions for the free and the NP-bound ToxRp in the presence of GdnHCl were 1.5 and 0.5 m respectively, whereas for urea denaturation, the values were 3.3 and 2.4 m, respectively. Far-UV CD spectra showed a significant change in the protein conformation upon binding to ZnO NPs, which was characterized by a substantial decrease in the α-helical content of the free protein. Isothermal titration calorimetry, used to quantify the thermodynamics of binding of ToxRp with ZnO NPs, showed an exothermic binding isotherm (ΔH = -9.8 kcal·mol(-1) and ΔS = -5.17 cal·mol(-1)·K(-1)).
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http://dx.doi.org/10.1111/j.1742-4658.2010.07807.xDOI Listing
October 2010

Structure and activity of lysozyme on binding to ZnO nanoparticles.

Langmuir 2010 Mar;26(5):3506-13

Department of Biochemistry, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054, India.

The interaction between ZnO nanoparticles (NPs) and lysozyme has been studied using calorimetric as well as spectrophotometric techniques, and interpreted in terms of the three-dimensional structure. The circular dichroism spectroscopic data show an increase in alpha-helical content on interaction with ZnO NPs. Glutaraldehyde cross-linking studies indicate that the monomeric form occurs to a greater extent than the dimer when lysozyme is conjugated with ZnO NPs. The enthalpy-driven binding between lysozyme and ZnO possibly involves the region encompassing the active site in the molecule, which is also the site for the dimer formation in a homologous structure. The enzyme retains high fraction of its native structure with negligible effect on its activity upon attachment to NPs. Compared to the free protein, lysozyme-ZnO conjugates are more stable in the presence of chaotropic agents (guanidine hydrochloride and urea) and also at elevated temperatures. The possible site of binding of NP to lysozyme has been proposed to explain these observations. The stability and the retention of a higher level of activity in the presence of the denaturing agent of the NP-conjugated protein may find useful applications in biotechnology ranging from diagnostic to drug delivery.
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http://dx.doi.org/10.1021/la903118cDOI Listing
March 2010