Papers by Dushyant K Garg
Aggregation of neuronal protein α-synuclein is implicated in synucleinopathies, including Parkins... more Aggregation of neuronal protein α-synuclein is implicated in synucleinopathies, including Parkinson’s disease. Despite abundant in vitro studies, the mechanism of α-synuclein assembly process remains ambiguous. In this work, α-synuclein aggregation was induced by its constant mixing in two separate modes, either by agitation in a 96-well microplate reader (MP) or in microcentrifuge tubes using a shaker incubator (SI). Aggregation in both modes occurred through a sigmoidal growth pattern with a well-defined lag, growth, and saturation phase. The end-stage MP- and SI-derived aggregates displayed distinct differences in morphological, biochemical, and spectral signatures as discerned through AFM, proteinase-K digestion, FTIR, Raman, and CD spectroscopy. The MP-derived aggregates showed irregular morphology with a significant random coil conformation, contrary to SI-derived aggregates, which showed typical β-sheet fibrillar structures. The end-stage MP aggregates convert to β-rich SI-li...
and assemble through strong intersubunit associative forces
International Journal of Biological Macromolecules, 2020
Chikungunya virus; the pathogen for chikungunya febrile and arthritic disease, having 11.8 kb pos... more Chikungunya virus; the pathogen for chikungunya febrile and arthritic disease, having 11.8 kb positive-sense RNA genome encodes polyproteins for structural and non-structural regions. The polyprotein (P1234) corresponding to the non-structural part from 5' end gets auto-cleaved by the action of nsP2 protease, which leads to the generation of individual functional enzymatic proteins like nsP4, nsP1, nsP2 and nsP3. Thus, nsP2 protein initiates viral replication. Targeting nsP2 to block virus replication has always been the foremost strategy to develop antivirals. Plant-based molecules are one of the top choices to develop as inhibitor due to their less toxicity and wide availability. Using a combination of receptor-based docking and MD simulations, we identified a flavanone glycoside- naringin, which binds to nsP2 protease at nM affinity. The biomolecular interaction between naringin and nsP2 was established through SPR. As discerned through FTIR and intrinsic fluorescence studies, upon binding with naringin, a global structural change in nsP2 occurs. This structural modulation in nsP2 due to binding of naringin is likely to interfere with the normal functioning of this enzyme during the viral life cycle. In conclusion, this report highlights the potential of naringin as an anti-viral agent against Chikungunya.
Journal of Biotechnology, 2020
Chaperones are a diverse class of molecules known for increasing thermo-stability of proteins, pr... more Chaperones are a diverse class of molecules known for increasing thermo-stability of proteins, preventing protein aggregation, favoring disaggregation, increasing solubility and in some cases imparting resistance to proteolysis. These functions can be employed for various biotechnological applications including point of care testing, nanobiotechnology, bio-process engineering, purification technologies and formulation development. Here we report that the N-terminal domain of Pyrococcus furiosus L-asparaginase, (NPfA, a protein chaperone lacking α-crystallin domain) can serve as an efficient, industrially relevant, protein additive. We tested the effect of NPfA on substrate proteins, ascorbate peroxidase (APX), IgG peroxidase antibodies (I-HAbs) and KOD DNA polymerase. Each protein not only displayed increased thermal stability but also increased activity in the presence of NPfA. This increase was either comparable or higher than those obtained by common osmolytes; glycine betaine, sorbitol and trehalose. Most dramatic activity enhancement was seen in the case of KOD polymerase (∼ 40 % increase). NPfA exerts its effect through transient binding to the substrate proteins as discerned through isothermal titration calorimetry, dynamic light scattering and size exclusion chromatography. Mechanistic insights obtained through simulations suggested a remodeled architecture and emergence of H-binding network between NPfA and substrate protein with an effective enhancement in the solvent accessibility at the active site pocket of the latter. Thus, the capability of NPfA to engage in specific manner with other proteins is demonstrated to reduce the concentration of substrate proteins/enzymes required per unit operation. The functional expansion obtained through our finding establishes NPfA as a novel class of ATP-independent molecular chaperone with immense future biotechnological applications.
Archives of Biochemistry and Biophysics, 2017
In obligate dimeric proteins of hyperthermophilic origin the question whether the native dimer is... more In obligate dimeric proteins of hyperthermophilic origin the question whether the native dimer is obtained by association of folded monomers or through concomitant folding and assembly of subunits has intrigued researchers. To find an answer we studied the folding of a dimeric enzyme L-asparaginase from Pyrococcus furiosus (PfA) for which we reported earlier that it unfolds cooperatively without populating folded monomeric intermediates. However, in the present study we report the finding of a folded monomeric intermediate of PfA under acidic condition. This monomer, although inactive, displayed secondary and tertiary structural features identical to the native protein and reassembled to active dimeric form upon reversal of pH. The monomer is conformationally flexible and thermodynamically and kinetically less stable than the native dimer. Interestingly, when incubated at 60 C the folded monomer, with exposed ANS-binding hydrophobic surfaces, spontaneously converted to amyloid fibrils. On the basis of our data we propose that PfA directly assembles into a multimeric form perhaps as an evolutionary adaptation to avoid accumulation of aggregation prone monomeric intermediates.
Biochimica et biophysica acta, Jan 2, 2016
Certain amino acid stretches are considered 'critical' to trigger amyloidogenesis in a pr... more Certain amino acid stretches are considered 'critical' to trigger amyloidogenesis in a protein. Synthetic peptides corresponding to these stretches are often used as experimental mimics for studying the amyloidogenesis of their parent protein. Here we provide evidence that such simple extrapolation is misleading. We scrutinized each step of amyloid progression of full length bovine carbonic anhydrase (BCA) and compared it with the amyloidogenic process of its critical peptide stretch 201-227 (PepB). We found that under similar solution conditions amyloidogenesis of BCA followed surface-catalyzed secondary nucleation, whereas, that of PepB followed classical nucleation-dependent pathway. AFM images showed that while BCA formed short, thick and branched fibrils, PepB formed thin, long and unbranched fibrils. Structural information obtained by ATR-FTIR spectroscopy suggested parallel arrangement of intermolecular β-sheet in BCA amyloids in contrast to PepB amyloids which arrang...
FEBS Journal, 2013
alpha-amylase and alpha-amylase bind by light scattering (View interaction) • Ab amyloid 1-42 and... more alpha-amylase and alpha-amylase bind by light scattering (View interaction) • Ab amyloid 1-42 and Ab amyloid 1-42 bind by transmission electron microscopy (View interaction) • NPfa binds to BCA II by anti tag coimmunoprecipitation (View interaction) • MSG and MSG bind by light scattering (View interaction) • BCA II and BCA II bind by light scattering (View interaction)
International journal of biological macromolecules, Jan 14, 2017
Heat shock proteins (HSPs) are known to confer protection to the stressed cells by rescuing vital... more Heat shock proteins (HSPs) are known to confer protection to the stressed cells by rescuing vital host cell proteins. In the present study we have demonstrated that heterologous expression of N-terminal domain of hyperthermophilic L-asparaginase (NPfA) confers thermotolerance to E. coli. The recombinant expression of NPfA enabled E. coli to demonstrate typical growth behavior at 52°C and survive a thermal shock up to 62°C, both being the highest reported temperatures for growth and heat shock survival. To understand the basis of protection proteome analysis of these cells was carried out which showed that NPfA guards a battery of proteins especially related to gene regulations and repair, providing definite survival advantage to the stressed cells. Thus, NPfA a non-canonical, non-natural chaperone has been shown to render E. coli cells with selective growth advantage under extremes of conditions. We propose that such modified, heat stabilized hosts could be utilized in developing he...
Heat shock proteins (HSPs) are known to confer protection to the stressed cells by rescuing vital... more Heat shock proteins (HSPs) are known to confer protection to the stressed cells by rescuing vital host cell proteins. In the present study we have demonstrated that heterologous expression of N-terminal domain of hyperthermophilic L-asparaginase (NPfA) confers thermotolerance to E. coli. The recombinant expression of NPfA enabled E. coli to demonstrate typical growth behavior at 52 • C and survive a thermal shock up to 62 • C, both being the highest reported temperatures for growth and heat shock survival. To understand the basis of protection proteome analysis of these cells was carried out which showed that NPfA guards a battery of proteins, especially related to gene regulations and repair, providing definite survival advantage to the stressed cells. Thus NPfA a non-canonical, non-natural chaperone has been shown to render E. coli cells with selective growth advantage under extremes of conditions. We propose that such modified, heat stabilized hosts could be utilized in developing heat-induced expression systems as well for the recombinant expression of thermophilic proteins.
In obligate dimeric proteins of hyperthermophilic origin the question whether the native dimer is... more In obligate dimeric proteins of hyperthermophilic origin the question whether the native dimer is obtained by association of folded monomers or through concomitant folding and assembly of subunits has intrigued researchers. To find an answer we studied the folding of a dimeric enzyme L-asparaginase from Pyrococcus furiosus (PfA) for which we reported earlier that it unfolds cooperatively without populating folded monomeric intermediates. However, in the present study we report the finding of a folded monomeric intermediate of PfA under acidic condition. This monomer, although inactive, displayed secondary and tertiary structural features identical to the native protein and reassembled to active dimeric form upon reversal of pH. The monomer is conformationally flexible and thermodynamically and kinetically less stable than the native dimer. Interestingly, when incubated at 60 C the folded monomer, with exposed ANS-binding hydrophobic surfaces, spontaneously converted to amyloid fibrils. On the basis of our data we propose that PfA directly assembles into a multimeric form perhaps as an evolutionary adaptation to avoid accumulation of aggregation prone monomeric intermediates.
Certain amino acid stretches are considered 'critical' to trigger amyloidogenesis in a protein. S... more Certain amino acid stretches are considered 'critical' to trigger amyloidogenesis in a protein. Synthetic peptides corresponding to these stretches are often used as experimental mimics for studying the amyloidogenesis of their parent protein. Here we provide evidence that such simple extrapolation is misleading. We scrutinized each step of amyloid progression of full length bovine carbonic anhydrase (BCA) and compared it with the amyloidogenic process of its critical peptide stretch 201–227 (PepB). We found that under similar solution conditions amyloidogenesis of BCA followed surface-catalyzed secondary nucleation, whereas, that of PepB followed classical nucleation-dependent pathway. AFM images showed that while BCA formed short, thick and branched fibrils, PepB formed thin, long and unbranched fibrils. Structural information obtained by ATR-FTIR spectroscopy suggested parallel arrangement of intermolecular β-sheet in BCA amyloids in contrast to PepB amyloids which arranged into antiparallel β sheets. Amyloids formed by BCA were unable to seed the fibrillation of PepB and vice versa. Even the intermediates formed during lag phase revealed contrasting FTIR and far UV CD signature, hy-drophobicity, morphology and cell cytotoxicity. Thus, we propose that sequences other than critical amyloidogenic stretches may significantly influence the initiation, polymerization and final fibrillar morphology of amyloid forming protein. The results have been discussed in light of primary sequence mediated amyloid polymorphism and its importance in the rational design of amyloid nanomaterials possessing desired physico-chemical properties.
Here, we report the folding and assembly of
a Pyrococcus furiosus-derived protein, l-asparaginase... more Here, we report the folding and assembly of
a Pyrococcus furiosus-derived protein, l-asparaginase
(PfA). PfA functions as a homodimer, with each monomer
made of distinct N- and C-terminal domains. The purified
individual domains as well as single Trp mutant of each
domain were subjected to chemical denaturation/renaturation
and probed by combination of spectroscopic, chromatographic,
quenching and scattering techniques. We found
that the N-domain acts like a folding scaffold and assists
the folding of remaining polypeptide. The domains displayed
sequential folding with the N-domain having higher
thermodynamic stability. We report that the extreme thermal
stability of PfA is due to the presence of high intersubunit
associative forces supported by extensive H-bonding
and ionic interactions network. Our results proved that
folding cooperativity in a thermophilic, multisubunit protein
is dictated by concomitant folding and association of
constituent domains directly into a native quaternary structure.
This report gives an account of the factors responsible
for folding and stability of a therapeutically and industrially
important protein.
• Ab amyloid 1-42 and Ab amyloid 1-42 bind by fluorescence technology (View interaction)
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Papers by Dushyant K Garg
a Pyrococcus furiosus-derived protein, l-asparaginase
(PfA). PfA functions as a homodimer, with each monomer
made of distinct N- and C-terminal domains. The purified
individual domains as well as single Trp mutant of each
domain were subjected to chemical denaturation/renaturation
and probed by combination of spectroscopic, chromatographic,
quenching and scattering techniques. We found
that the N-domain acts like a folding scaffold and assists
the folding of remaining polypeptide. The domains displayed
sequential folding with the N-domain having higher
thermodynamic stability. We report that the extreme thermal
stability of PfA is due to the presence of high intersubunit
associative forces supported by extensive H-bonding
and ionic interactions network. Our results proved that
folding cooperativity in a thermophilic, multisubunit protein
is dictated by concomitant folding and association of
constituent domains directly into a native quaternary structure.
This report gives an account of the factors responsible
for folding and stability of a therapeutically and industrially
important protein.
a Pyrococcus furiosus-derived protein, l-asparaginase
(PfA). PfA functions as a homodimer, with each monomer
made of distinct N- and C-terminal domains. The purified
individual domains as well as single Trp mutant of each
domain were subjected to chemical denaturation/renaturation
and probed by combination of spectroscopic, chromatographic,
quenching and scattering techniques. We found
that the N-domain acts like a folding scaffold and assists
the folding of remaining polypeptide. The domains displayed
sequential folding with the N-domain having higher
thermodynamic stability. We report that the extreme thermal
stability of PfA is due to the presence of high intersubunit
associative forces supported by extensive H-bonding
and ionic interactions network. Our results proved that
folding cooperativity in a thermophilic, multisubunit protein
is dictated by concomitant folding and association of
constituent domains directly into a native quaternary structure.
This report gives an account of the factors responsible
for folding and stability of a therapeutically and industrially
important protein.