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Shridhar K Sathe - One of the best experts on this subject based on the ideXlab platform.
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interactions with 8 anilinonaphthalene 1 sulfonic Acid ans and surface hydrophobicity of black gram vigna mungo phaseolin
Journal of Food Science, 2018Co-Authors: Maithili Deshpande, Shridhar K SatheAbstract:Surface hydrophobicity (SH) properties of the trimeric storage protein phaseolin (black gram phaseolin [BGP]) of black gram (Vigna mungo) were investigated using 8‐anilinonaphthalene‐1‐sulfonate (ANS) as an extrinsic fluorescent probe. The emission maxima of fluorescence spectra of BGP:ANS complex were blue‐shifted to 455 nm as compared to 515 nm for the free ANS. Saturation binding occurred at a dye‐to‐protein ratio of about 30:1. The quantum yield of the complex increased with increasing ionic strength. The Kdvalues were 1.7 × 10−5and 1.37 × 10−5M using fractional occupancy and Scatchard analysis, respectively. Analysis of the binding data using Klotz plot revealed 4 binding sites/protomer. SH of BGP was 48%, which rapidly decreased due to the perturbation of the binding sites as the protein unfolded in GdnHCl and urea. By varying processing conditions, it may be possible to alter the surface exposure of SH of BGP to extend its applications in novel food products with desired textural attributes. Varying solvent and/or processing conditions can assist to modulate the surface hydrophobicity of functional legume proteins to achieve desired textural properties in the end product.
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Interactions with 8‐Anilinonaphthalene‐1‐sulfonic Acid (ANS) and Surface Hydrophobicity of Black Gram (Vigna mungo) Phaseolin
Journal of food science, 2018Co-Authors: Maithili Deshpande, Shridhar K SatheAbstract:Surface hydrophobicity (SH) properties of the trimeric storage protein phaseolin (black gram phaseolin [BGP]) of black gram (Vigna mungo) were investigated using 8‐anilinonaphthalene‐1‐sulfonate (ANS) as an extrinsic fluorescent probe. The emission maxima of fluorescence spectra of BGP:ANS complex were blue‐shifted to 455 nm as compared to 515 nm for the free ANS. Saturation binding occurred at a dye‐to‐protein ratio of about 30:1. The quantum yield of the complex increased with increasing ionic strength. The Kdvalues were 1.7 × 10−5and 1.37 × 10−5M using fractional occupancy and Scatchard analysis, respectively. Analysis of the binding data using Klotz plot revealed 4 binding sites/protomer. SH of BGP was 48%, which rapidly decreased due to the perturbation of the binding sites as the protein unfolded in GdnHCl and urea. By varying processing conditions, it may be possible to alter the surface exposure of SH of BGP to extend its applications in novel food products with desired textural attributes. Varying solvent and/or processing conditions can assist to modulate the surface hydrophobicity of functional legume proteins to achieve desired textural properties in the end product.
Maithili Deshpande - One of the best experts on this subject based on the ideXlab platform.
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interactions with 8 anilinonaphthalene 1 sulfonic Acid ans and surface hydrophobicity of black gram vigna mungo phaseolin
Journal of Food Science, 2018Co-Authors: Maithili Deshpande, Shridhar K SatheAbstract:Surface hydrophobicity (SH) properties of the trimeric storage protein phaseolin (black gram phaseolin [BGP]) of black gram (Vigna mungo) were investigated using 8‐anilinonaphthalene‐1‐sulfonate (ANS) as an extrinsic fluorescent probe. The emission maxima of fluorescence spectra of BGP:ANS complex were blue‐shifted to 455 nm as compared to 515 nm for the free ANS. Saturation binding occurred at a dye‐to‐protein ratio of about 30:1. The quantum yield of the complex increased with increasing ionic strength. The Kdvalues were 1.7 × 10−5and 1.37 × 10−5M using fractional occupancy and Scatchard analysis, respectively. Analysis of the binding data using Klotz plot revealed 4 binding sites/protomer. SH of BGP was 48%, which rapidly decreased due to the perturbation of the binding sites as the protein unfolded in GdnHCl and urea. By varying processing conditions, it may be possible to alter the surface exposure of SH of BGP to extend its applications in novel food products with desired textural attributes. Varying solvent and/or processing conditions can assist to modulate the surface hydrophobicity of functional legume proteins to achieve desired textural properties in the end product.
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Interactions with 8‐Anilinonaphthalene‐1‐sulfonic Acid (ANS) and Surface Hydrophobicity of Black Gram (Vigna mungo) Phaseolin
Journal of food science, 2018Co-Authors: Maithili Deshpande, Shridhar K SatheAbstract:Surface hydrophobicity (SH) properties of the trimeric storage protein phaseolin (black gram phaseolin [BGP]) of black gram (Vigna mungo) were investigated using 8‐anilinonaphthalene‐1‐sulfonate (ANS) as an extrinsic fluorescent probe. The emission maxima of fluorescence spectra of BGP:ANS complex were blue‐shifted to 455 nm as compared to 515 nm for the free ANS. Saturation binding occurred at a dye‐to‐protein ratio of about 30:1. The quantum yield of the complex increased with increasing ionic strength. The Kdvalues were 1.7 × 10−5and 1.37 × 10−5M using fractional occupancy and Scatchard analysis, respectively. Analysis of the binding data using Klotz plot revealed 4 binding sites/protomer. SH of BGP was 48%, which rapidly decreased due to the perturbation of the binding sites as the protein unfolded in GdnHCl and urea. By varying processing conditions, it may be possible to alter the surface exposure of SH of BGP to extend its applications in novel food products with desired textural attributes. Varying solvent and/or processing conditions can assist to modulate the surface hydrophobicity of functional legume proteins to achieve desired textural properties in the end product.
Samrat Mukhopadhyay - One of the best experts on this subject based on the ideXlab platform.
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Dynamics and dimension of an amyloidogenic disordered state of human β_2-microglobulin
European Biophysics Journal, 2013Co-Authors: Dominic Narang, Pushpender K. Sharma, Samrat MukhopadhyayAbstract:Human β_2-microglobulin (β_2m) aggregation is implicated in dialysis-related amyloidosis. Previously, it has been shown that β_2m adopts an ensemble of partially unfolded states at low pH. Here we provide detailed structural and dynamical insights into the Acid unfolded and yet compact state of β_2m at pH 2.5 using a host of fluorescence spectroscopic tools. These tools allowed us to investigate protein conformational dynamics at low micromolar protein concentrations in an amyloid-forming condition. Our equilibrium fluorescence data in combination with circular dichroism data provide support in favor of progressive structural dissolution of β_2m with lowering pH. The Acid unfolded intermediate at pH 2.5 has high 8-anilinonaphthalene, 1-sulfonic Acid (ANS)-binding affinity and is devoid of significant secondary structural elements. Using fluorescence lifetime measurements, we have been able to monitor the conformational transition during the pH transition from the native to the compact disordered state. Additionally, using time-resolved fluorescence anisotropy measurements, we have been able to distinguish this compact disordered state from the canonical denatured state of the protein by identifying unique dynamic signatures pertaining to the segmental chain mobility. Taken together, our results demonstrate that β_2m at pH 2.5 adopts a compact noncanonical unfolded state resembling a collapsed premolten globule state. Additionally, our stopped-flow fluorescence kinetics results provide mechanistic insights into the formation of a compact disordered state from the native form.
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Dynamics and dimension of an amyloidogenic disordered state of human β2-microglobulin
European biophysics journal : EBJ, 2013Co-Authors: Dominic Narang, Pushpender K. Sharma, Samrat MukhopadhyayAbstract:Human β2-microglobulin (β2m) aggregation is implicated in dialysis-related amyloidosis. Previously, it has been shown that β2m adopts an ensemble of partially unfolded states at low pH. Here we provide detailed structural and dynamical insights into the Acid unfolded and yet compact state of β2m at pH 2.5 using a host of fluorescence spectroscopic tools. These tools allowed us to investigate protein conformational dynamics at low micromolar protein concentrations in an amyloid-forming condition. Our equilibrium fluorescence data in combination with circular dichroism data provide support in favor of progressive structural dissolution of β2m with lowering pH. The Acid unfolded intermediate at pH 2.5 has high 8-anilinonaphthalene, 1-sulfonic Acid (ANS)-binding affinity and is devoid of significant secondary structural elements. Using fluorescence lifetime measurements, we have been able to monitor the conformational transition during the pH transition from the native to the compact disordered state. Additionally, using time-resolved fluorescence anisotropy measurements, we have been able to distinguish this compact disordered state from the canonical denatured state of the protein by identifying unique dynamic signatures pertaining to the segmental chain mobility. Taken together, our results demonstrate that β2m at pH 2.5 adopts a compact noncanonical unfolded state resembling a collapsed premolten globule state. Additionally, our stopped-flow fluorescence kinetics results provide mechanistic insights into the formation of a compact disordered state from the native form.
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Kinetics of Surfactant-induced Aggregation of Lysozyme Studied by Fluorescence Spectroscopy
Journal of Fluorescence, 2011Co-Authors: Neha Jain, Mily Bhattacharya, Samrat MukhopadhyayAbstract:The study of protein conformational changes in the presence of surfactants and lipids is important in the context of protein folding and misfolding. In the present study, we have investigated the mechanism of the protein conformational change coupled with aggregation leading to size growth of Hen Egg White Lysozyme (HEWL) in the presence of an anionic detergent such as sodium dodecyl sulphate (SDS) in alkaline pH. We have utilized intrinsic protein fluorescence (tryptophan) and extrinsic fluorescent reporters such as 8-Anilinonaphthalene-1-Sulfonic Acid (ANS), dansyl and fluorescein to follow the protein conformational change in real-time. By analyzing the kinetics of fluorescence intensity and anisotropy of multiple fluorescent reporters, we have been able to delineate the mechanism of surfactant-induced aggregation of lysozyme. The kinetic parameters reveal that aggregation proceeds with an initial fast-phase (conformational change) followed by a slow-phase (self-assembly). Our results indicate that SDS, below critical micelle concentration, induces conformational expansion that triggers the aggregation process at a micromolar protein concentration range.
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Structure and dynamics of a molecular hydrogel derived from a tripodal cholamide.
Journal of the American Chemical Society, 2004Co-Authors: Samrat Mukhopadhyay, Uday Maitra, Ira, Guruswamy Krishnamoorthy, Judith Schmidt, Yeshayahu TalmonAbstract:Tripodal cholamide 1 is a supergelator of aqueous fluids. A variety of physical techniques, including cryo-transmission electron microscopy (TEM), circular dichroism (CD), steady-state fluorescence, time-resolved fluorescence, and dynamic light-scattering, were employed to understand the structure and dynamics of the gel. Fluorescent probes [ANS (8-Anilinonaphthalene-1-Sulfonic Acid) and pyrene] reported two critical aggregation concentrations $(CAC_1 and CAC_2)$ of 1 in predominantly aqueous media, with the minimum gel concentration (MGC) being close to $CAC_2$. Fluorescence lifetime measurements with pyrene revealed ineffective quenching of pyrene fluorescence by oxygen, possibly caused by slower Brownian diffusion due to the enhanced viscosity in the gel phase. The study of the gelation kinetics by monitoring the ultrafast dynamics of ANS revealed a progressive increase in the aggregate size and the microviscosity of the aqueous pool encompassed by the self-assembled fibrillar network (SAFIN) during the gelation. The striking difference between microviscosity and bulk (macroscopic) viscosity of the gel is also discussed.lifetime measurements with pyrenerevealed ineffective quenching of pyrene fluorescence by oxygen,possibly caused by slower Brownian diffusion due to the enhancedviscosity in the gel phase. The study of the gelation kinetics bymonitoring the ultrafast dynamics of ANS revealed a progressiveincrease in the aggregate size and the microviscosity of the aqueouspool encompassed by the self-assembled fibrillar network (SAFIN) duringthe gelation. The striking difference between microviscosity and bulk(macroscopic) viscosity of the gel is also discussed.
Ashok Kumar Mishra - One of the best experts on this subject based on the ideXlab platform.
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Application of Partial Least Square (PLS) Analysis on Fluorescence Data of 8-Anilinonaphthalene-1-Sulfonic Acid, a Polarity Dye, for Monitoring Water Adulteration in Ethanol Fuel
Journal of Fluorescence, 2015Co-Authors: Keshav Kumar, Ashok Kumar MishraAbstract:Fluorescence characteristic of 8-Anilinonaphthalene-1-Sulfonic Acid (ANS) in ethanol-water mixture in combination with partial least square (PLS) analysis was used to propose a simple and sensitive analytical procedure for monitoring the adulteration of ethanol by water. The proposed analytical procedure was found to be capable of detecting even small adulteration level of ethanol by water. The robustness of the procedure is evident from the statistical parameters such as square of correlation coefficient (R^2), root mean square of calibration (RMSEC) and root mean square of prediction (RMSEP) that were found to be well with in the acceptable limits.
Rizwan Hasan Khan - One of the best experts on this subject based on the ideXlab platform.
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Unravelling the inhibitory and cytoprotective potential of diuretics towards amyloid fibrillation.
International journal of biological macromolecules, 2019Co-Authors: Sadia Malik, Mohammad Khursheed Siddiqi, Aiman Masroor, Nabeela Majid, Syed Moasfar Ali, Rizwan Hasan KhanAbstract:Abstract Protein misfolding and deposition of aggregated proteins inside as well as outside of the cells have been associated with several neurotoxic and neurodegenerative disorders like Alzheimer’s, Parkinson’s and familial amyloid polyneuropathy etc. that could be controlled by anti-aggregation methodologies employing either inhibition or disaggregation of toxic aggregates. Also, the Alzheimer’s disease develops in later life is somehow related to the high mid-life blood pressure. Therefore the present work targets the amyloid inhibiting potential of diuretics (a class of antihypertensive drugs) – Indapamide (INDP) and Hydrochlorothiazide (HCTZ) against human serum albumin (HSA) and human lysozyme (HL) fibrillogenesis. The effect of both INDP and HCTZ on the kinetics of amyloid formation of HSA and HL was illustrated and various biophysical techniques like Thioflavin T (ThT) and 8-Anilinonaphthalene-1-Sulfonic Acid (ANS) fluorescence measurement, Congo red measurements and circular dichroism (CD) measurements depicted the inhibitory action of both INDP and HCTZ in a dose dependent manner. Transmission Electronic Microscopy (TEM) confirmed the absence of fibrillar structures when HSA and HL were co-incubated with INDP and HCTZ. In addition, molecular docking results revealed that both the drugs interacts with HSA and HL through hydrophobic interactions as well as hydrogen bonding, and also showed non-hemolytic activity on human RBCs demonstrated by the Hemolytic assay. Thus, both INDP and HCTZ could be propitious as a therapeutic agent and aid in the cure of amyloid related diseases.
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Deciphering the enhanced inhibitory, disaggregating and cytoprotective potential of promethazine towards amyloid fibrillation.
International journal of biological macromolecules, 2017Co-Authors: Saima Nusrat, Masihuz Zaman, Aiman Masroor, Mohammad Khursheed Siddqi, Nida Zaidi, Km Neelofar, Ali S. Abdelhameed, Rizwan Hasan KhanAbstract:Abstract Increasing evidence proposed that amyloid deposition by proteins play a crucial role in an array of neurotoxic and degenerative disorders like Parkinson’s disease, systemic amyloidosis etc, that could be controlled by anti-aggregation methodologies which either inhibit or disaggregate such toxic aggregates. The present work targets the amyloid inhibiting and disaggregating potential of promethazine (PRM) against human insulin (HI) and human lysozyme (HL) fibrillogenesis. Biophysical techniques like Rayleigh scattering measurements (RLS), Thioflavin T (ThT) and 8-Anilinonaphthalene-1-Sulfonic Acid (ANS) fluorescence measurement, circular dichroism (CD) and dynamic light scattering (DLS) measurements illustrated the inhibitory action of PRM. The half maximal inhibitory concentration (IC 50 ) of PRM for HI and HL was estimated to be 114.81 ± 1.21 μM and 186.20 ± 1.03 μM, respectively. Microscopic techniques revealed the absence of fibrillar structures when HI and HL was co-incubated with PRM. Cytoprotective behavior of PRM was investigated by cell based cytotoxicity assay performed on SH-SY5Y neuronal cell lines. The half maximal disaggregation concentration (DC 50 ) was calculated as 21.37 ± 0.89 μM and 45.70 ± 0.76 μM, signifying that PRM is much potent to disaggregate pre formed fibrils rather than to inhibit fibrillation. Thus, PRM could be beneficial as therapeutic agent that can aid in the cure of amyloid related diseases.
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Effect of surfactants on Ra-sHSPI – A small heat shock protein from the cattle tick Rhipicephalus annulatus
Journal of Molecular Structure, 2016Co-Authors: Mohammad Khursheed Siddiqi, Yasser E. Shahein, Nahla A. Hussein, Rizwan Hasan KhanAbstract:Abstract Electrostatic interaction plays an important role in protein aggregation phenomenon. In this study, we have checked the effect of anionic – Sodium Dodecyl Sulfate (SDS) and cationic-Cetyltrimethyl Ammonium Bromide (CTAB) surfactant on aggregation behavior of Ra-sHSPI, a small heat shock protein purified from Rhipicephalus annulatus tick. To monitor the effect of these surfactants, we have employed several spectroscopic methods such as Rayleigh light scattering measurements, ANS (8-Anilinonaphthalene-1-Sulfonic Acid) fluorescence measurements, ThT (Thioflavin T) binding assays, Far-UV CD (Circular Dichroism) and dynamic light scattering measurements. In the presence of anionic surfactant-SDS, Ra-sHSPI forms amyloid fibrils, in contrast, no amyloid formation was observed in presence of cationic surfactant at low pH. Enhancement of ANS fluorescence intensity confirms the exposition of more hydrophobic patches during aggregation. ThT binding assay confirms the amyloid fibrillar nature of the SDS induced Ra-sHSPI aggregates and supported by PASTA 2.0 (prediction of amyloid structural aggregation) software. This study demonstrates the crucial role of charge during amyloid fibril formation at low pH in Ra-sHSPI.
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phytolacca americana lectin pa 2 pokeweed mitogen an intrinsically unordered protein and its conversion into partial order at low ph
Bioscience Reports, 2009Co-Authors: Ejaz Ahmad, Shah Kamranur Rahman, Javed Masood Khan, Ankita Varshney, Rizwan Hasan KhanAbstract:: This is the first report of its kind that well demonstrates that a lectin from Phytolacca americana [Pa-2 (P. americana lectin-2)] can also be intrinsically unordered, based on the results obtained by CD, tryptophan fluorescence, ANS (8-Anilinonaphthalene-1-Sulfonic Acid) binding, acrylamide quenching, DLS (dynamic light scattering) and its amino Acid composition database analyses. Pa-2 is an Acidic monomeric lectin and acquires random coil conformation at neutral pH without any regular secondary structure. As confirmed by different spectroscopic techniques, on lowering the pH, some secondary structures, predominantly alpha-helices, are detected by far-UV CD that adopt a marginally stable partially folded collapsed conformation possessing the characteristics of a premolten globule state. It is in accordance with coil-helix transition that is commonly observed when these intrinsically unordered proteins interact with their partner molecules in vivo.
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Fluoroalcohols induced unfolding of succinylated Con A : Native like β-structure in partially folded intermediate and α-helix in molten globule like state
Archives of biochemistry and biophysics, 2006Co-Authors: Sadaf Fatima, Basir Ahmad, Rizwan Hasan KhanAbstract:Abstract Concanavalin A (Con A) exists in dimeric state at pH 5. In concentration range 20–60% (v/v) 2,2,2-trifluoroethanol (TFE) and 2–40% (v/v) 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), Con A at pH 5.0 shows visible aggregation. However, when succinyl Con A was used, no aggregation was observed in the entire concentration range of fluoroalcohols (0–90% v/v TFE and HFIP) and resulted in stable α-helix formation. Temperature-induced concentration-dependent aggregation in Con A was also found to be prevented/reduced in succinylated form. Possible role of electrostatic repulsion among residues in the prevention of hydrophobically driven aggregation has been discussed. Results indicate that succinylation of a protein resulted in greater stability (in both β-sheet and α-helical forms) against alcohol-induced and temperature-induced concentration-dependent aggregation and this observation may play significant role in amyloid-forming proteins. Effect of TFE and HFIP on the conformation of a dimeric protein, Succinylated Con A, has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of hydrophobic dye ANS (8-Anilinonaphthalene-1-Sulfonic Acid). Far UV-CD, a probe for secondary structure shows loss of native secondary structure in the presence of low concentration of both the alcohols, TFE (10% v/v) and HFIP (4% v/v). Upon addition of higher concentration of these alcohols, Succinylated Con A exhibited transformation from β-sheet to α-helical structure. Intrinsic tryptophan fluorescence studies, ANS binding and near UV-CD experiments indicate the protein is more expanded, have more exposed hydrophobic surfaces and highly disrupted tertiary structure at 60% (v/v) TFE and 30% (v/v) HFIP concentrations. Taken together, these results it might be concluded that TFE and HFIP induce two intermediate states at their low and high concentrations in Succinyl Con A.
