Rajagopal Subramanyam - Academia.edu (original) (raw)

Papers by Rajagopal Subramanyam

Research paper thumbnail of Elucidation of binding mechanism of stigmasterol with human serum albumin: a biophysical and molecular dynamics simulation approach

Journal of Biomolecular Structure and Dynamics

In the present study, we have analyzed the interaction of a phytochemical, stigmasterol (Stig), w... more In the present study, we have analyzed the interaction of a phytochemical, stigmasterol (Stig), with human serum albumin (HSA) under physiological conditions using fluorescence quenching, circular dichroism and molecular modeling methods. Cytotoxic studies with Stig in mouse macrophages (RAW 246.7) and HeLa cell lines showed anti-inflammatory and anti-cancer properties. Further, the intrinsic fluorescence of HSA was quenched by Stig, which was considered a static quenching mechanism. The site-specific marker experiments revealed that Stig binds to the IIIA subdomain of HSA with a binding constant of KStig=1.8 ± 0.03 × 105 M-1 and free energy of -7.26 ± 0.031 Kcal/mol. The secondary structure of HSA was partially unfolded after binding of Stig, which indicates an alteration in the microenvironment of the protein binding site. Molecular docking experiments found that Stig binds strongly with HSA at the IIIA domain of the hydrophobic pocket with one hydrogen bond. The rigidity of the protein-Stig complex and free energies were analyzed by molecular dynamic simulation (MDS) for 100 ns, where the HSA-Stig was stabilized after 40 ns. MDS studies revealed that HSA does not significantly change the secondary structure when it binds with Stig, which is in agreement with the circular dichroism data. Overall, the results obtained gave qualitative and quantitative insight into the binding interaction between HSA and Stig, which is essential in understanding the latter as a therapeutic molecule.Communicated by Ramaswamy H. Sarma.

Research paper thumbnail of Relationship between the redox state of P700 and variable thermal dissipation in isolated photosystem I particles

The relationship between the redox state of P700 monitored using absorbance changes at 830 nm and... more The relationship between the redox state of P700 monitored using absorbance changes at 830 nm and photochemical energy storage in PSI reaction centers assayed with the photoacoustic method (PA) was studied in isolated PSI submembrane particles aspirated onto nitrocellulose filters. Several donors have been used to support the electron transport through PSI. NADPH and NADH demonstrated low rates of electron donation to PSI, while ascorbate and ascorbate plus 2,6-dichlorophenolindophenol (DCIP) couple have been found more effective in both P700+ reduction and stimulation of the variable component of the PA signal. A linear relationship was found in isolated PSI particles between the (A830, max - A830, steady)/A830, max and (PAmax - PAsteady)/PAmax ratios, which characterized the relative amount of P700 in the reduced state and the relative magnitude of the variable PA component, respectively. That linear relationship was independent on the nature of electron donor used for the reducti...

Research paper thumbnail of Nature and mechanism of the light-induced reversible structural changes of LHCII

Research paper thumbnail of Deciphering the AChE-binding mechanism with multifunctional tricyclic coumarin anti-Alzheimer's agents using biophysical and bioinformatics approaches and evaluation of their modulating effect on Amyloidogenic peptide assembly

International Journal of Biological Macromolecules

Investigating the drug-AChE binding mechanism is vital in understanding its cogent use in medical... more Investigating the drug-AChE binding mechanism is vital in understanding its cogent use in medical practice against Alzheimer's disease (AD). The production and accumulation of oligomers of β-amyloid is a central event in the neuropathology of AD. Beside the inhibition of assembly process, modulation of the aggregation process of these proteins towards minimally toxic pathways may be a possible therapeutic strategy for AD. Hence, the present study aims to examine the effect of multifunctional fused tricyclic 7-hydroxy 4-methy coumarin analogs (HMC1-5) on the self-induced aggregation of β-amyloid using Thioflavin T (ThT) assay, scanning electron microscopic study, AlamarBlue and immune blotting assays and also the binding mechanism with AChE by fluorescence emission, conformational, molecular docking and molecular dynamic simulation studies under physiological pH 7.4. The ThT assay, FE-SEM study, cell line and western blots establish that the HMC1-5 molecules could irreversibly disrupt preformed Aβ42 fibrils, accelerate the aggregates into micro size co-assembled structures, and effectively eliminate the cytotoxicity of Aβ1-42. Fluorescence emission studies indicating a strong binding affinity between HMC1-5 and AChE with the binding constants of 1.04 × 105, 3.57 × 104, 1.97 × 104, 3.07 × 104 and 2.95 × 104 M-1, respectively and binding sites number found to be 1. CD studies disclosed a partial unfolding in the secondary structure of AChE upon binding with HMC1-5. Docking analysis inferred that the HMC1-5 were bound through hydrophobic and hydrophilic interactions to the AChE active site. Molecular dynamics simulations emphasized the stability of AChE-HMC1-5 complexes throughout the 100 ns simulations, and the local conformational changes of the residues of AChE validate the stability of complexes. These results provide new and unique complementary approach for modulating the biological effects of the Aβ aggregates by coumarin analogs and new insights for further in vivo investigations as novel anti AD agents.

Research paper thumbnail of Improving the inhibition of β-amyloid aggregation by withanolide and withanoside derivatives

International Journal of Biological Macromolecules

Here, we have studied the ameliorative effects of Withania somnifera derivatives (Withanolide A, ... more Here, we have studied the ameliorative effects of Withania somnifera derivatives (Withanolide A, Withanolide B, Withanoside IV, and Withanoside V) on the fibril formation of amyloid-β 42 for Alzheimer's disease. We analyzed reduction in the aggregation of β amyloid protein with these Ashwagandha derivatives by Thioflavin T assay in the oligomeric and fibrillar state. We have tested the cytotoxic activity of these compounds against human SK-N-SH cell line for 48 h, and the IC 50 value found to be 28.61 ± 2.91, 14.84 ± 1.45, 18.76 ± 0.76 and 30.14 ± 2.59 μM respectively. After the treatment of the cells with half the concentration of IC 50 value, there was a remarkable decrease in the number of apoptotic cells stained by TUNEL assay indicating the DNA damage and also observed significant decrease of reactive oxygen species. Also, the binding and molecular stability of these derivatives with amyloid β was also studied using bioinformatics tools where these molecules were interacted at LVFFA region which is inhibition site of amyloid-β 42. These studies revealed that the Withanolides and Withanosides interact with the hydrophobic core of amyloid-β 42 in the oligomeric stage, preventing further interaction with the monomers and diminishing aggregation.

Research paper thumbnail of Protection of Energy Transfer Process in Isolated Phycobilisome by "White Light" from Ultraviolet-B Induced Damage in the Cyanobacterium Spirulina Platensis

Research paper thumbnail of Comparative binding of Swertiamarin with human serum albumin and α-1 glycoprotein and its cytotoxicity against neuroblastoma cells

Journal of Biomolecular Structure and Dynamics

Communicated by Ramaswamy H. Sarma

Research paper thumbnail of Autophagy induced accumulation of lipids in pgrl1 and pgr5 of Chlamydomonas reinhardtii under high light

Chlamydomonas (C) reinhardtii cells (wild-type CC125 and 137AH, and cyclic electron transport dep... more Chlamydomonas (C) reinhardtii cells (wild-type CC125 and 137AH, and cyclic electron transport dependant mutants pgrl1 and pgr5) were grown in high light 500 μmol photons m-2 s-1 where the growth was significantly enhanced after three days. The starch and lipid contents were also increased; however, starch content was decreased in pgr5. Further, the Nile Red fluorescence shows that a significant amount of lipid bodies were observed in pgr5 cells under high light. Similarly, the electron micrographs show that large vacuoles were formed in high light stress despite the change in stacks of grana structure. We also observed increased production of reactive oxygen species (ROS) that could lead to autophagy. Inline, a significant increase of ATG8 protein was noticed in pgr5, which is a hallmark characteristic for autophagy formation. Consequently, the triacylglycerol (TAG) content was increased due to DGAT and PDAT enzymes' expression, especially in pgr5. Here, the TAG synthesis would ...

Research paper thumbnail of Role of cyclic electron transport mutations pgrl1 and pgr5 in acclimation process to high light in Chlamydomonas reinhardtii

Photosynthesis Research

Light is crucial for photosynthesis, but the amount of light that exceeds an organism’s assimilat... more Light is crucial for photosynthesis, but the amount of light that exceeds an organism’s assimilation efficacy can lead to photo-oxidative damage and even cell death. In Chlamydomonas ( C ). reinhardtii cyclic electron flow (CEF) is very important for the elicitation of non-photochemical quenching (NPQ) by controlling the acidification of thylakoid lumen. This process requires the cooperation of proton gradient regulation (PGR) proteins, PGRL1 and PGR5. Here, we compared the growth pattern and photosynthetic activity between wild type ( 137c , t222 +) and mutants impaired in CEF ( pgrl1 and pgr5 ) under photoautotrophic and photoheterotrophic conditions. We have observed the discriminative expression of NPQ in the mutants impaired in CEF of pgrl1 and pgr5. The results obtained from the mutants showed reduced cell growth and density, Chl a / b ratio, fluorescence, electron transport rate, and yield of photosystem (PS)II. These mutants have reduced capability to develop a strong NPQ indicating that the role of CEF is very crucial for photoprotection. Moreover, the CEF mutant exhibits increased photosensitivity compared with the wild type. Therefore, we suggest that besides NPQ, the fraction of non-regulated non-photochemical energy loss (NO) also plays a crucial role during high light acclimation despite a low growth rate. This low NPQ rate may be due to less influx of protons coming from the CEF in cases of pgrl1 and pgr5 mutants. These results are discussed in terms of the relative photoprotective benefit, related to the thermal dissipation of excess light in photoautotrophic and photoheterotrophic conditions.

Research paper thumbnail of International Conference on “Photosynthesis and Hydrogen Energy Research for Sustainability-2017”

Research paper thumbnail of Changes in the photosynthetic apparatus and lipid droplet formation in Chlamydomonas reinhardtii under iron deficiency

Photosynthesis Research

The unicellular photosynthetic alga Chlamydomonas reinhardtii was propagated in iron deficiency m... more The unicellular photosynthetic alga Chlamydomonas reinhardtii was propagated in iron deficiency medium and patterns of growth, photosynthetic efficiency, lipid accumulation, as well as the expression of lipid biosynthetic and photosynthesis-related proteins were analysed and compared with iron-sufficient growth conditions. As expected, the photosynthetic rate was reduced (maximally after 4 days of growth) as a result of increased non-photochemical quenching (NPQ). Surprisingly, the stress-response protein LHCSR3 was expressed in conditions of iron deficiency that cause NPQ induction. In addition, the protein contents of both the PSI and PSII reaction centres were gradually reduced during growth in iron deficiency medium. Interestingly, the two generations of Fe deficiency cells could be able to recover the photosynthesis but the second generation cells recovered much slower as these cells were severely in shock. Analysis by flow cytometry with fluorescence-activated cell sorting and thin layer chromatography showed that iron deficiency also induced the accumulation of triacylglycerides (TAG), which resulted in the formation of lipid droplets. This was most significant between 48 and 72 h of growth. Dramatic increases in DGAT2A and PDAT1 levels were caused by iron starvation, which indicated that the biosynthesis of TAG had been increased. Analysis using gas chromatography mass spectrometry showed that levels of 16:0, 18:0, 18:2 and 18:3Δ9,12,15 fatty acids were significantly elevated. The results of this study highlight the genes/enzymes of Chlamydomonas that affect lipid synthesis through their influence on photosynthesis, and these represent potential targets of metabolic engineering to develop strains for biofuel production.

Research paper thumbnail of Unraveling the stability of plasma proteins upon interaction of synthesized uridine products: biophysical and molecular dynamics approach

Journal of Biomolecular Structure and Dynamics

Abstract Most of the drugs binding to human serum albumin (HSA) are transported to various parts ... more Abstract Most of the drugs binding to human serum albumin (HSA) are transported to various parts of the body. Here, we have studied the molecular interaction between HSA and synthesized uridine derivatives, 1-[(3R, 4S, 5 R)-2-methyl-3, 4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidine-2,4-dion.)(C-MU); [(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxy-4-methyl-tetrahydrofuran-2-yl] methyl methyl phosphochloridate (CM-MU) and [(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-2-methyl-3,4-dihydroxyoxolan-2-yl] methyl dihydrogen phosphate (P-MU). Cytotoxic studies of these synthesized compounds with mouse macrophages (RAW 246.7) and HeLa cells (human cervical cancer cells) and binding mechanism of these uridine derivatives with HSA were performed. Subsequently, fluorescence quenching was observed upon titration of uridine derivatives with HSA via static mode of quenching, and the binding constants (K2-C-MU = 4 ± 0.03 × 104M−1, K5-CM-MU = 1.95 ± 0.03 × 104 M−1 and K5-P-MU =1.56 ± 0.03 × 104 M−1) were found to be in sync with the computational results. Further, molecular displacement and molecular docking data revealed that all the derivatives are binding in the subdomain IIA and IIB regions of HSA. The protein secondary structure of complexes was determined by circular dichroism, indicating partial unfolding of the protein upon addition of the uridine derivatives. Furthermore, atomic force microscopy data reveal the change in topology upon binding of 2-C-MU, 5-CM-MU and 5-P-MU with HSA, indicating change in the microenvironment around tryptophan region. Additionally, cytotoxicity studies on HeLa and Raw Cell lines suggested that these molecules have significant anti-proliferative and anti-inflammatory properties. Hence, the study may be of help for development of new drugs based on uridine derivatives which may be helpful for combating various potential diseases. Communicated by Ramaswamy H. Sarma

Research paper thumbnail of Role of Herborn (K240E) and Milano Slow (D375H) human serum albumin variants towards binding of phenylbutazone and ibuprofen

International Journal of Biological Macromolecules

Human serum albumin (HSA) is the binding cargo in blood plasma. The binding of drugs to HSA deter... more Human serum albumin (HSA) is the binding cargo in blood plasma. The binding of drugs to HSA determines the pharmacokinetics and pharmacodynamics of the drugs. There are 67 natural genetic variants of HSA were reported in literature. Studying the effect of albumin modifications on drug binding helps to treat the patients with proper medication. In the present study, we have aimed to understand the effect of two natural variants of HSA, such as Herborn (K240E) and Milano Slow (D375H) on the binding of phenylbutazone and ibuprofen. For this, we have generated K240E and D375H mutants and also double mutant (K240E/D375H) of HSA using site directed mutagenesis. The recombinant HSA and its variants were expressed in Pichia pastoris. The interaction of HSA and its variants to phenylbutazone and ibuprofen was studied using fluorescence spectroscopy. Our results showed that there is no significant effect of K240E and D375H mutations on phenylbutazone and ibuprofen binding. But the effect is significant when both the mutations were there in a single protein (K240E/D375H). Further, the CD spectroscopy data showed that there is no effect of phenylbutazone and ibuprofen binding on the conformation of protein, except in case of D375H, where there is a conformational change in the binding pocket with the ibuprofen binding.

Research paper thumbnail of Forskolin loaded human serum albumin nanoparticles and its biological importance

Journal of Biomolecular Structure and Dynamics

Abstract In this study, forskolin-loaded human serum albumin nanoparticles (FR-HSANPs) were succe... more Abstract In this study, forskolin-loaded human serum albumin nanoparticles (FR-HSANPs) were successfully prepared by incorporation and affinity-binding methods. FR-HSANPs were characterized by transmission electron microscope that most of them are circular in shape and size is around 340 nm. The drug loading was more than 88% and further sustained release profiles were observed as it is 77.5% in 24 h time. Additionally, the cytotoxicity results with HepG2 cells indicated that FR-HSANPs showed significantly higher cytotoxicity and lower cell viability as compared to free forskolin (FR). Furthermore, to understand the binding mechanism of human serum albumin (HSA) with forskolin resulted from fluorescence quenching as a static mechanism and the binding constant is 6.26 ± 0.1 × 104 M−1, indicating a strong binding affinity. Further, association and dissociation kinetics of forskolin–HSA was calculated from surface plasmon resonance spectroscopy and the binding constant found to be Kforskolin = 3.4 ± 0.24 × 104 M−1 and also fast dissociation was observed. Further, we used circular dichroism and molecular dynamics simulations to elucidate the possible structural changes including local conformational changes and rigidity of the residues of both HSA and HSA–forskolin complexes. Communicated by Ramaswamy H. Sarma

Research paper thumbnail of Synthesis and biological evaluation of flavone-8-acrylamide derivatives as potential multi-target-directed anti Alzheimer agents and investigation of binding mechanism with acetylcholinesterase

Bioorganic Chemistry

In a search for novel multifunctional anti-Alzheimer agents, a congeneric set of seventeen flavon... more In a search for novel multifunctional anti-Alzheimer agents, a congeneric set of seventeen flavone-8-acrylamide derivatives (8a─q) were synthesized and evaluated for their cholinesterase inhibitory, antioxidant, neuroprotective and modulation of Aβ aggregation activities. The target compounds showed effective and selective inhibitory activity against the AChE over BuChE. In addition, the target compounds also showed moderate anti-oxidant activity and strong neuroprotective capacities, and accelerated dosage-dependently the Aβ aggregation. Also, we presented here a complete study on the interaction of 8a, 8d, 8e, 8h and 8i with AChE. Through fluorescence emission studies, the binding sites number found to be 1, binding constants were calculated as 2.04 × 104, 2.22 × 104, 1.18 × 104, 9.8 × 103 and 3.2 × 104 M-1 and free energy change as -5.83, -5.91, -5.51, -5.41 and -6.12 kcal M-1 at 25 °C which were well agreed with the computational calculations indicating a strong binding affinity of flavones and AChE. Furthermore, the CD studies revealed that the secondary structure of AChE became partly unfolded upon binding with 8a, 8d, 8e, 8h and 8i.

Research paper thumbnail of Non-photochemical quenching-dependent acclimation and thylakoid organization of Chlamydomonas reinhardtii to high light stress

Photosynthesis Research

Light is essential for all photosynthetic organisms while an excess of it can lead to damage main... more Light is essential for all photosynthetic organisms while an excess of it can lead to damage mainly the photosystems of the thylakoid membrane. In this study, we have grown Chlamydomonas reinhardtii cells in different intensities of high light to understand the photosynthetic process with reference to thylakoid membrane organization during its acclimation process. We observed, the cells acclimatized to long-term response to high light intensities of 500 and 1000 µmol m−2 s−1 with faster growth and more biomass production when compared to cells at 50 µmol m−2 s−1 light intensity. The ratio of Chl a/b was marginally decreased from the mid-log phase of growth at the high light intensity. Increased level of zeaxanthin and LHCSR3 expression was also found which is known to play a key role in non-photochemical quenching (NPQ) mechanism for photoprotection. Changes in photosynthetic parameters were observed such as increased levels of NPQ, marginal change in electron transport rate, and many other changes which demonstrate that cells were acclimatized to high light which is an adaptive mechanism. Surprisingly, PSII core protein contents have marginally reduced when compared to peripherally arranged LHCII in high light-grown cells. Further, we also observed alterations in stromal subunits of PSI and low levels of PsaG, probably due to disruption of PSI assembly and also its association with LHCI. During the process of acclimation, changes in thylakoid organization occurred in high light intensities with reduction of PSII supercomplex formation. This change may be attributed to alteration of protein–pigment complexes which are in agreement with circular dichoism spectra of high light-acclimatized cells, where decrease in the magnitude of psi-type bands indicates changes in ordered arrays of PSII–LHCII supercomplexes. These results specify that acclimation to high light stress through NPQ mechanism by expression of LHCSR3 and also observed changes in thylakoid protein profile/supercomplex formation lead to low photochemical yield and more biomass production in high light condition.

Research paper thumbnail of Exploration of binding studies of β-oxalyldiamino propionic acid (β-ODAP), a non-protein amino acid with human serum albumin-biophysical and computational approach

Journal of Biomolecular Structure and Dynamics

Communicated by Ramaswamy H. Sarma

Research paper thumbnail of Elucidating the active interaction mechanism of phytochemicals withanolide and withanoside derivatives with human serum albumin

Research paper thumbnail of Honoring eight senior distinguished plant biologists from India

Photosynthesis research, Jan 14, 2018

We summarize here research contributions of eight stalwarts in photosynthesis research from India... more We summarize here research contributions of eight stalwarts in photosynthesis research from India. These distinguished scientists (Shree Kumar Apte, Basanti Biswal, Udaya C. Biswal, Agepati S. Raghavendra, Attipalli Ramachandra Reddy, Prafullachandra Vishnu (Raj) Sane, Baishnab Charan Tripathy, and Dinesh C. Uprety) were honored on November 2, 2017, at the School of Life Sciences, University of Hyderabad. We include here two group photographs of this special event, which was organized by the Department of Plant Sciences, during the 8th International Conference on Photosynthesis and Hydrogen Energy Research for Sustainability-2017 ( https://prs.science/wp-content/uploads/2017/10/Photosynthesis-Research-for-Sustainability-2017.pdf , also available at: http://www.life.illinois.edu/govindjee/world-historical.html ). The main conference had honored three international scientists: William Cramer (Purdue University. West Lafayette, Indiana, USA), Govindjee (University of Illinois at Urbana...

Research paper thumbnail of Unraveling the Stability of Plasma Proteins upon Interaction of Synthesized Androstenedione and Its Derivatives-A Biophysical and Computational Approach

ACS omega, Jan 31, 2017

4-Androstene-3-17-dione (4A), also known as androstenedione, is the key intermediate of steroid m... more 4-Androstene-3-17-dione (4A), also known as androstenedione, is the key intermediate of steroid metabolism. 5β-Androstane-3-17-dione (5A) and (+)-6-methyl-5β-androstane-3-17-dione (6M) are the steroid derivatives of androstenedione. The interactions of androstenedione and its derivatives with plasma proteins are important in understanding the distribution and bioavailability of these molecules. In our present study, we have studied the binding affinity of androstenedione and its derivatives with plasma proteins such as human serum albumin (HSA) and α1-acid glycoprotein (AGP). Our results showed that the 4A, 5A, and 6M steroid molecules can form stable complexes with HSA and AGP. The affinity of the studied steroid molecules with HSA is high compared to that with AGP, and the binding constants obtained for 4A, 5A, and 6M with HSA are 5.3 ± 2 × 10, 5.3 ± 1 × 10, and 9.5 ± 0.2 × 10 M, respectively. Further, binding sites of these steroid molecules in HSA are identified using molecular ...

Research paper thumbnail of Elucidation of binding mechanism of stigmasterol with human serum albumin: a biophysical and molecular dynamics simulation approach

Journal of Biomolecular Structure and Dynamics

In the present study, we have analyzed the interaction of a phytochemical, stigmasterol (Stig), w... more In the present study, we have analyzed the interaction of a phytochemical, stigmasterol (Stig), with human serum albumin (HSA) under physiological conditions using fluorescence quenching, circular dichroism and molecular modeling methods. Cytotoxic studies with Stig in mouse macrophages (RAW 246.7) and HeLa cell lines showed anti-inflammatory and anti-cancer properties. Further, the intrinsic fluorescence of HSA was quenched by Stig, which was considered a static quenching mechanism. The site-specific marker experiments revealed that Stig binds to the IIIA subdomain of HSA with a binding constant of KStig=1.8 ± 0.03 × 105 M-1 and free energy of -7.26 ± 0.031 Kcal/mol. The secondary structure of HSA was partially unfolded after binding of Stig, which indicates an alteration in the microenvironment of the protein binding site. Molecular docking experiments found that Stig binds strongly with HSA at the IIIA domain of the hydrophobic pocket with one hydrogen bond. The rigidity of the protein-Stig complex and free energies were analyzed by molecular dynamic simulation (MDS) for 100 ns, where the HSA-Stig was stabilized after 40 ns. MDS studies revealed that HSA does not significantly change the secondary structure when it binds with Stig, which is in agreement with the circular dichroism data. Overall, the results obtained gave qualitative and quantitative insight into the binding interaction between HSA and Stig, which is essential in understanding the latter as a therapeutic molecule.Communicated by Ramaswamy H. Sarma.

Research paper thumbnail of Relationship between the redox state of P700 and variable thermal dissipation in isolated photosystem I particles

The relationship between the redox state of P700 monitored using absorbance changes at 830 nm and... more The relationship between the redox state of P700 monitored using absorbance changes at 830 nm and photochemical energy storage in PSI reaction centers assayed with the photoacoustic method (PA) was studied in isolated PSI submembrane particles aspirated onto nitrocellulose filters. Several donors have been used to support the electron transport through PSI. NADPH and NADH demonstrated low rates of electron donation to PSI, while ascorbate and ascorbate plus 2,6-dichlorophenolindophenol (DCIP) couple have been found more effective in both P700+ reduction and stimulation of the variable component of the PA signal. A linear relationship was found in isolated PSI particles between the (A830, max - A830, steady)/A830, max and (PAmax - PAsteady)/PAmax ratios, which characterized the relative amount of P700 in the reduced state and the relative magnitude of the variable PA component, respectively. That linear relationship was independent on the nature of electron donor used for the reducti...

Research paper thumbnail of Nature and mechanism of the light-induced reversible structural changes of LHCII

Research paper thumbnail of Deciphering the AChE-binding mechanism with multifunctional tricyclic coumarin anti-Alzheimer's agents using biophysical and bioinformatics approaches and evaluation of their modulating effect on Amyloidogenic peptide assembly

International Journal of Biological Macromolecules

Investigating the drug-AChE binding mechanism is vital in understanding its cogent use in medical... more Investigating the drug-AChE binding mechanism is vital in understanding its cogent use in medical practice against Alzheimer's disease (AD). The production and accumulation of oligomers of β-amyloid is a central event in the neuropathology of AD. Beside the inhibition of assembly process, modulation of the aggregation process of these proteins towards minimally toxic pathways may be a possible therapeutic strategy for AD. Hence, the present study aims to examine the effect of multifunctional fused tricyclic 7-hydroxy 4-methy coumarin analogs (HMC1-5) on the self-induced aggregation of β-amyloid using Thioflavin T (ThT) assay, scanning electron microscopic study, AlamarBlue and immune blotting assays and also the binding mechanism with AChE by fluorescence emission, conformational, molecular docking and molecular dynamic simulation studies under physiological pH 7.4. The ThT assay, FE-SEM study, cell line and western blots establish that the HMC1-5 molecules could irreversibly disrupt preformed Aβ42 fibrils, accelerate the aggregates into micro size co-assembled structures, and effectively eliminate the cytotoxicity of Aβ1-42. Fluorescence emission studies indicating a strong binding affinity between HMC1-5 and AChE with the binding constants of 1.04 × 105, 3.57 × 104, 1.97 × 104, 3.07 × 104 and 2.95 × 104 M-1, respectively and binding sites number found to be 1. CD studies disclosed a partial unfolding in the secondary structure of AChE upon binding with HMC1-5. Docking analysis inferred that the HMC1-5 were bound through hydrophobic and hydrophilic interactions to the AChE active site. Molecular dynamics simulations emphasized the stability of AChE-HMC1-5 complexes throughout the 100 ns simulations, and the local conformational changes of the residues of AChE validate the stability of complexes. These results provide new and unique complementary approach for modulating the biological effects of the Aβ aggregates by coumarin analogs and new insights for further in vivo investigations as novel anti AD agents.

Research paper thumbnail of Improving the inhibition of β-amyloid aggregation by withanolide and withanoside derivatives

International Journal of Biological Macromolecules

Here, we have studied the ameliorative effects of Withania somnifera derivatives (Withanolide A, ... more Here, we have studied the ameliorative effects of Withania somnifera derivatives (Withanolide A, Withanolide B, Withanoside IV, and Withanoside V) on the fibril formation of amyloid-β 42 for Alzheimer's disease. We analyzed reduction in the aggregation of β amyloid protein with these Ashwagandha derivatives by Thioflavin T assay in the oligomeric and fibrillar state. We have tested the cytotoxic activity of these compounds against human SK-N-SH cell line for 48 h, and the IC 50 value found to be 28.61 ± 2.91, 14.84 ± 1.45, 18.76 ± 0.76 and 30.14 ± 2.59 μM respectively. After the treatment of the cells with half the concentration of IC 50 value, there was a remarkable decrease in the number of apoptotic cells stained by TUNEL assay indicating the DNA damage and also observed significant decrease of reactive oxygen species. Also, the binding and molecular stability of these derivatives with amyloid β was also studied using bioinformatics tools where these molecules were interacted at LVFFA region which is inhibition site of amyloid-β 42. These studies revealed that the Withanolides and Withanosides interact with the hydrophobic core of amyloid-β 42 in the oligomeric stage, preventing further interaction with the monomers and diminishing aggregation.

Research paper thumbnail of Protection of Energy Transfer Process in Isolated Phycobilisome by "White Light" from Ultraviolet-B Induced Damage in the Cyanobacterium Spirulina Platensis

Research paper thumbnail of Comparative binding of Swertiamarin with human serum albumin and α-1 glycoprotein and its cytotoxicity against neuroblastoma cells

Journal of Biomolecular Structure and Dynamics

Communicated by Ramaswamy H. Sarma

Research paper thumbnail of Autophagy induced accumulation of lipids in pgrl1 and pgr5 of Chlamydomonas reinhardtii under high light

Chlamydomonas (C) reinhardtii cells (wild-type CC125 and 137AH, and cyclic electron transport dep... more Chlamydomonas (C) reinhardtii cells (wild-type CC125 and 137AH, and cyclic electron transport dependant mutants pgrl1 and pgr5) were grown in high light 500 μmol photons m-2 s-1 where the growth was significantly enhanced after three days. The starch and lipid contents were also increased; however, starch content was decreased in pgr5. Further, the Nile Red fluorescence shows that a significant amount of lipid bodies were observed in pgr5 cells under high light. Similarly, the electron micrographs show that large vacuoles were formed in high light stress despite the change in stacks of grana structure. We also observed increased production of reactive oxygen species (ROS) that could lead to autophagy. Inline, a significant increase of ATG8 protein was noticed in pgr5, which is a hallmark characteristic for autophagy formation. Consequently, the triacylglycerol (TAG) content was increased due to DGAT and PDAT enzymes' expression, especially in pgr5. Here, the TAG synthesis would ...

Research paper thumbnail of Role of cyclic electron transport mutations pgrl1 and pgr5 in acclimation process to high light in Chlamydomonas reinhardtii

Photosynthesis Research

Light is crucial for photosynthesis, but the amount of light that exceeds an organism’s assimilat... more Light is crucial for photosynthesis, but the amount of light that exceeds an organism’s assimilation efficacy can lead to photo-oxidative damage and even cell death. In Chlamydomonas ( C ). reinhardtii cyclic electron flow (CEF) is very important for the elicitation of non-photochemical quenching (NPQ) by controlling the acidification of thylakoid lumen. This process requires the cooperation of proton gradient regulation (PGR) proteins, PGRL1 and PGR5. Here, we compared the growth pattern and photosynthetic activity between wild type ( 137c , t222 +) and mutants impaired in CEF ( pgrl1 and pgr5 ) under photoautotrophic and photoheterotrophic conditions. We have observed the discriminative expression of NPQ in the mutants impaired in CEF of pgrl1 and pgr5. The results obtained from the mutants showed reduced cell growth and density, Chl a / b ratio, fluorescence, electron transport rate, and yield of photosystem (PS)II. These mutants have reduced capability to develop a strong NPQ indicating that the role of CEF is very crucial for photoprotection. Moreover, the CEF mutant exhibits increased photosensitivity compared with the wild type. Therefore, we suggest that besides NPQ, the fraction of non-regulated non-photochemical energy loss (NO) also plays a crucial role during high light acclimation despite a low growth rate. This low NPQ rate may be due to less influx of protons coming from the CEF in cases of pgrl1 and pgr5 mutants. These results are discussed in terms of the relative photoprotective benefit, related to the thermal dissipation of excess light in photoautotrophic and photoheterotrophic conditions.

Research paper thumbnail of International Conference on “Photosynthesis and Hydrogen Energy Research for Sustainability-2017”

Research paper thumbnail of Changes in the photosynthetic apparatus and lipid droplet formation in Chlamydomonas reinhardtii under iron deficiency

Photosynthesis Research

The unicellular photosynthetic alga Chlamydomonas reinhardtii was propagated in iron deficiency m... more The unicellular photosynthetic alga Chlamydomonas reinhardtii was propagated in iron deficiency medium and patterns of growth, photosynthetic efficiency, lipid accumulation, as well as the expression of lipid biosynthetic and photosynthesis-related proteins were analysed and compared with iron-sufficient growth conditions. As expected, the photosynthetic rate was reduced (maximally after 4 days of growth) as a result of increased non-photochemical quenching (NPQ). Surprisingly, the stress-response protein LHCSR3 was expressed in conditions of iron deficiency that cause NPQ induction. In addition, the protein contents of both the PSI and PSII reaction centres were gradually reduced during growth in iron deficiency medium. Interestingly, the two generations of Fe deficiency cells could be able to recover the photosynthesis but the second generation cells recovered much slower as these cells were severely in shock. Analysis by flow cytometry with fluorescence-activated cell sorting and thin layer chromatography showed that iron deficiency also induced the accumulation of triacylglycerides (TAG), which resulted in the formation of lipid droplets. This was most significant between 48 and 72 h of growth. Dramatic increases in DGAT2A and PDAT1 levels were caused by iron starvation, which indicated that the biosynthesis of TAG had been increased. Analysis using gas chromatography mass spectrometry showed that levels of 16:0, 18:0, 18:2 and 18:3Δ9,12,15 fatty acids were significantly elevated. The results of this study highlight the genes/enzymes of Chlamydomonas that affect lipid synthesis through their influence on photosynthesis, and these represent potential targets of metabolic engineering to develop strains for biofuel production.

Research paper thumbnail of Unraveling the stability of plasma proteins upon interaction of synthesized uridine products: biophysical and molecular dynamics approach

Journal of Biomolecular Structure and Dynamics

Abstract Most of the drugs binding to human serum albumin (HSA) are transported to various parts ... more Abstract Most of the drugs binding to human serum albumin (HSA) are transported to various parts of the body. Here, we have studied the molecular interaction between HSA and synthesized uridine derivatives, 1-[(3R, 4S, 5 R)-2-methyl-3, 4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidine-2,4-dion.)(C-MU); [(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxy-4-methyl-tetrahydrofuran-2-yl] methyl methyl phosphochloridate (CM-MU) and [(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-2-methyl-3,4-dihydroxyoxolan-2-yl] methyl dihydrogen phosphate (P-MU). Cytotoxic studies of these synthesized compounds with mouse macrophages (RAW 246.7) and HeLa cells (human cervical cancer cells) and binding mechanism of these uridine derivatives with HSA were performed. Subsequently, fluorescence quenching was observed upon titration of uridine derivatives with HSA via static mode of quenching, and the binding constants (K2-C-MU = 4 ± 0.03 × 104M−1, K5-CM-MU = 1.95 ± 0.03 × 104 M−1 and K5-P-MU =1.56 ± 0.03 × 104 M−1) were found to be in sync with the computational results. Further, molecular displacement and molecular docking data revealed that all the derivatives are binding in the subdomain IIA and IIB regions of HSA. The protein secondary structure of complexes was determined by circular dichroism, indicating partial unfolding of the protein upon addition of the uridine derivatives. Furthermore, atomic force microscopy data reveal the change in topology upon binding of 2-C-MU, 5-CM-MU and 5-P-MU with HSA, indicating change in the microenvironment around tryptophan region. Additionally, cytotoxicity studies on HeLa and Raw Cell lines suggested that these molecules have significant anti-proliferative and anti-inflammatory properties. Hence, the study may be of help for development of new drugs based on uridine derivatives which may be helpful for combating various potential diseases. Communicated by Ramaswamy H. Sarma

Research paper thumbnail of Role of Herborn (K240E) and Milano Slow (D375H) human serum albumin variants towards binding of phenylbutazone and ibuprofen

International Journal of Biological Macromolecules

Human serum albumin (HSA) is the binding cargo in blood plasma. The binding of drugs to HSA deter... more Human serum albumin (HSA) is the binding cargo in blood plasma. The binding of drugs to HSA determines the pharmacokinetics and pharmacodynamics of the drugs. There are 67 natural genetic variants of HSA were reported in literature. Studying the effect of albumin modifications on drug binding helps to treat the patients with proper medication. In the present study, we have aimed to understand the effect of two natural variants of HSA, such as Herborn (K240E) and Milano Slow (D375H) on the binding of phenylbutazone and ibuprofen. For this, we have generated K240E and D375H mutants and also double mutant (K240E/D375H) of HSA using site directed mutagenesis. The recombinant HSA and its variants were expressed in Pichia pastoris. The interaction of HSA and its variants to phenylbutazone and ibuprofen was studied using fluorescence spectroscopy. Our results showed that there is no significant effect of K240E and D375H mutations on phenylbutazone and ibuprofen binding. But the effect is significant when both the mutations were there in a single protein (K240E/D375H). Further, the CD spectroscopy data showed that there is no effect of phenylbutazone and ibuprofen binding on the conformation of protein, except in case of D375H, where there is a conformational change in the binding pocket with the ibuprofen binding.

Research paper thumbnail of Forskolin loaded human serum albumin nanoparticles and its biological importance

Journal of Biomolecular Structure and Dynamics

Abstract In this study, forskolin-loaded human serum albumin nanoparticles (FR-HSANPs) were succe... more Abstract In this study, forskolin-loaded human serum albumin nanoparticles (FR-HSANPs) were successfully prepared by incorporation and affinity-binding methods. FR-HSANPs were characterized by transmission electron microscope that most of them are circular in shape and size is around 340 nm. The drug loading was more than 88% and further sustained release profiles were observed as it is 77.5% in 24 h time. Additionally, the cytotoxicity results with HepG2 cells indicated that FR-HSANPs showed significantly higher cytotoxicity and lower cell viability as compared to free forskolin (FR). Furthermore, to understand the binding mechanism of human serum albumin (HSA) with forskolin resulted from fluorescence quenching as a static mechanism and the binding constant is 6.26 ± 0.1 × 104 M−1, indicating a strong binding affinity. Further, association and dissociation kinetics of forskolin–HSA was calculated from surface plasmon resonance spectroscopy and the binding constant found to be Kforskolin = 3.4 ± 0.24 × 104 M−1 and also fast dissociation was observed. Further, we used circular dichroism and molecular dynamics simulations to elucidate the possible structural changes including local conformational changes and rigidity of the residues of both HSA and HSA–forskolin complexes. Communicated by Ramaswamy H. Sarma

Research paper thumbnail of Synthesis and biological evaluation of flavone-8-acrylamide derivatives as potential multi-target-directed anti Alzheimer agents and investigation of binding mechanism with acetylcholinesterase

Bioorganic Chemistry

In a search for novel multifunctional anti-Alzheimer agents, a congeneric set of seventeen flavon... more In a search for novel multifunctional anti-Alzheimer agents, a congeneric set of seventeen flavone-8-acrylamide derivatives (8a─q) were synthesized and evaluated for their cholinesterase inhibitory, antioxidant, neuroprotective and modulation of Aβ aggregation activities. The target compounds showed effective and selective inhibitory activity against the AChE over BuChE. In addition, the target compounds also showed moderate anti-oxidant activity and strong neuroprotective capacities, and accelerated dosage-dependently the Aβ aggregation. Also, we presented here a complete study on the interaction of 8a, 8d, 8e, 8h and 8i with AChE. Through fluorescence emission studies, the binding sites number found to be 1, binding constants were calculated as 2.04 × 104, 2.22 × 104, 1.18 × 104, 9.8 × 103 and 3.2 × 104 M-1 and free energy change as -5.83, -5.91, -5.51, -5.41 and -6.12 kcal M-1 at 25 °C which were well agreed with the computational calculations indicating a strong binding affinity of flavones and AChE. Furthermore, the CD studies revealed that the secondary structure of AChE became partly unfolded upon binding with 8a, 8d, 8e, 8h and 8i.

Research paper thumbnail of Non-photochemical quenching-dependent acclimation and thylakoid organization of Chlamydomonas reinhardtii to high light stress

Photosynthesis Research

Light is essential for all photosynthetic organisms while an excess of it can lead to damage main... more Light is essential for all photosynthetic organisms while an excess of it can lead to damage mainly the photosystems of the thylakoid membrane. In this study, we have grown Chlamydomonas reinhardtii cells in different intensities of high light to understand the photosynthetic process with reference to thylakoid membrane organization during its acclimation process. We observed, the cells acclimatized to long-term response to high light intensities of 500 and 1000 µmol m−2 s−1 with faster growth and more biomass production when compared to cells at 50 µmol m−2 s−1 light intensity. The ratio of Chl a/b was marginally decreased from the mid-log phase of growth at the high light intensity. Increased level of zeaxanthin and LHCSR3 expression was also found which is known to play a key role in non-photochemical quenching (NPQ) mechanism for photoprotection. Changes in photosynthetic parameters were observed such as increased levels of NPQ, marginal change in electron transport rate, and many other changes which demonstrate that cells were acclimatized to high light which is an adaptive mechanism. Surprisingly, PSII core protein contents have marginally reduced when compared to peripherally arranged LHCII in high light-grown cells. Further, we also observed alterations in stromal subunits of PSI and low levels of PsaG, probably due to disruption of PSI assembly and also its association with LHCI. During the process of acclimation, changes in thylakoid organization occurred in high light intensities with reduction of PSII supercomplex formation. This change may be attributed to alteration of protein–pigment complexes which are in agreement with circular dichoism spectra of high light-acclimatized cells, where decrease in the magnitude of psi-type bands indicates changes in ordered arrays of PSII–LHCII supercomplexes. These results specify that acclimation to high light stress through NPQ mechanism by expression of LHCSR3 and also observed changes in thylakoid protein profile/supercomplex formation lead to low photochemical yield and more biomass production in high light condition.

Research paper thumbnail of Exploration of binding studies of β-oxalyldiamino propionic acid (β-ODAP), a non-protein amino acid with human serum albumin-biophysical and computational approach

Journal of Biomolecular Structure and Dynamics

Communicated by Ramaswamy H. Sarma

Research paper thumbnail of Elucidating the active interaction mechanism of phytochemicals withanolide and withanoside derivatives with human serum albumin

Research paper thumbnail of Honoring eight senior distinguished plant biologists from India

Photosynthesis research, Jan 14, 2018

We summarize here research contributions of eight stalwarts in photosynthesis research from India... more We summarize here research contributions of eight stalwarts in photosynthesis research from India. These distinguished scientists (Shree Kumar Apte, Basanti Biswal, Udaya C. Biswal, Agepati S. Raghavendra, Attipalli Ramachandra Reddy, Prafullachandra Vishnu (Raj) Sane, Baishnab Charan Tripathy, and Dinesh C. Uprety) were honored on November 2, 2017, at the School of Life Sciences, University of Hyderabad. We include here two group photographs of this special event, which was organized by the Department of Plant Sciences, during the 8th International Conference on Photosynthesis and Hydrogen Energy Research for Sustainability-2017 ( https://prs.science/wp-content/uploads/2017/10/Photosynthesis-Research-for-Sustainability-2017.pdf , also available at: http://www.life.illinois.edu/govindjee/world-historical.html ). The main conference had honored three international scientists: William Cramer (Purdue University. West Lafayette, Indiana, USA), Govindjee (University of Illinois at Urbana...

Research paper thumbnail of Unraveling the Stability of Plasma Proteins upon Interaction of Synthesized Androstenedione and Its Derivatives-A Biophysical and Computational Approach

ACS omega, Jan 31, 2017

4-Androstene-3-17-dione (4A), also known as androstenedione, is the key intermediate of steroid m... more 4-Androstene-3-17-dione (4A), also known as androstenedione, is the key intermediate of steroid metabolism. 5β-Androstane-3-17-dione (5A) and (+)-6-methyl-5β-androstane-3-17-dione (6M) are the steroid derivatives of androstenedione. The interactions of androstenedione and its derivatives with plasma proteins are important in understanding the distribution and bioavailability of these molecules. In our present study, we have studied the binding affinity of androstenedione and its derivatives with plasma proteins such as human serum albumin (HSA) and α1-acid glycoprotein (AGP). Our results showed that the 4A, 5A, and 6M steroid molecules can form stable complexes with HSA and AGP. The affinity of the studied steroid molecules with HSA is high compared to that with AGP, and the binding constants obtained for 4A, 5A, and 6M with HSA are 5.3 ± 2 × 10, 5.3 ± 1 × 10, and 9.5 ± 0.2 × 10 M, respectively. Further, binding sites of these steroid molecules in HSA are identified using molecular ...