Biology subjects

Bandyopadhyay, P.

Publications and source records attributed to Bandyopadhyay, P..

6 recordsLinked to original sources

bioRxiv2024

Calcium Binding Affinity in the Mutational Landscape of Troponin-C: Free Energy Calculation, Co-evolution modeling and Machine Learning

Mutation in calcium-binding proteins (CBPs) can significantly influence Ca2+ binding affinity (BA), resulting in substantial impairment in the signaling process and leading to several lethal diseases. The knowledge behind the changes in the binding affinity can help in understanding the signaling process and designing inhibitors for therapeutic usage. However, accurate prediction of BA for a large number of mutations has been elusive. In this work, for an important calcium binding protein, cardiac Troponin-C, we have developed an integrative modeling approach that combines molecular dynamics (MD)-based binding free energy calculations, prediction of plausible mutants using evolutionary information, and an interpretable machine learning model to predict Ca2+ BA for a large number of mutations (seventy-six in all). For the binding free energy calculation, we have used a charge-scaling based MD simulation that considers the polarization in the system, which is critical for divalent ion binding with proteins. The well-known molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method was used for the binding free energy calculations. The calculated results for twenty-four disease mutants, which are associated with different cardiomyopathies and have experimental binding affinity, are in close agreement with the experimental results. To study other plausible mutations, we have probed the evolutionary landscape of cardiac Troponin-C and used the EVmutation method of Hopf et al.(Nature biotechnology 2017, 35, 128-135) to generate sixty-one additional mutants. Finally, a Support vector regression model was developed for both observed and plausible mutations. Our machine learning model used simple structure and sequence-based descriptors along with MD-based descriptors and gave a mean squared error (MSE) of only 0.16 kcal/mol. Assessment of the contribution of each descriptor shows that the number of water molecules within the Ca2+ binding site, type of amino acid substitution (e.g. polar to hydrophobic reduces the binding affinity), and the distance of mutation with Ca2+ are the most important factors in determining the binding affinity. This integrative modeling can be used for other CBPs and can lay the path for modeling the complex and astronomically large mutational landscape of Calcium-binding proteins.

biophysics↗

bioRxiv2024

Understanding Cu<+2> binding with DNA: A molecular dynamics study comparing Cu<2+> and Mg<2+> binding to the Dickerson DNA

Cu2+ ions led DNA damage by reactive oxygen species (ROS) is widely known biological phenomena. The ionic radii of Cu2+ and Mg2+ being similar, the binding of Cu2+ ions to DNA is expected to be similar to that of the Mg2+ ions. However, little is known how Cu2+ ions bind in different parts (phosphate, major and minor grooves) of a double-strand (ds) DNA, especially at atomic level. In the present study, we employ molecular dynamic (MD) simulations to investigate the binding of Cu2+ ions with the Dickerson DNA, a B-type dodecamer double stranded (ds) DNA. The binding characteristics of Cu2+ and Mg2+ ions with this dsDNA are compared to get an insight into the differences and similarities in binding behavior of both ions. Unlike Mg2+ ions, the first hydration shell of Cu2+ is found to be labile, thus it shows both direct and indirect binding with the dsDNA, i.e., binding through displacement of water from the hydration shell or through the hydration shell. Though the binding propensity of Cu2+ ions with dsDNA is observed relatively stronger, the binding order to phosphates, major groove, and minor groove is found qualitatively similar (phosphates > major groove > minor groove) for both ions. The study gives a deep understanding of Cu2+ binding to DNA, which could be helpful in rationalizing the Cu2+ led ROS-mediated DNA damage.

biophysics↗

bioRxiv2024

Chloroquine attenuates hypoxia-mediated autophagy to curb thrombosis- an ex vivo and in vivo study

BackgroundHypoxia can trigger the activation of blood platelets, leading to thrombosis. If not addressed clinically, it can cause severe complications and fatal consequences as well. The current treatment regime for thrombosis is often palliative and includes long-term administration of anticoagulants, which has the risk of over-bleeding in injury and other secondary effects as well. This demands a deeper understanding of the process and exploration of an alternative therapeutic avenue. Interestingly, recent studies demonstrate that platelets though atypical and enucleated, possess components of autophagy machinery. This cellular homeostatic process though well-studied in non-platelet cells, is under-explored in platelets. MethodsIn this study, we report an activation of autophagy in rat-derived platelets cultured under physiologically relevant hypoxic condition (10% O2) ex vivo. Furthermore, autophagy was triggered in vivo when rats were exposed to hypobaric hypoxic conditions. Subsequently, restriction or surgical ligation of the inferior vena cava (IVC) was performed to induce thrombus formation. Post confirming the impact of autophagy induction on platelet functioning, it was inhibited, and then platelet activation and aggregation status were evaluated using light transmission aggregometry, flow cytometry, immunofluorescence and immunoblotting. ResultsHerein, we show that autophagy inhibition with the potent autophagy inhibitor-CQ, a repurposed FDA-approved drug, can significantly reduce platelet activation, both in ex vivo and in vivo settings. CQ withdrawal reversed the phenomenon indicating a dynamic effect. Thereafter, in flow restriction or surgical ligation model, interestingly, CQ-pre-treated rats showed reduced clotting ability. Importantly, CQ at the stipulated dose was found to be non-toxic to the tissues, as analyzed through histological staining. ConclusionThus, we propose that the repurpose of the FDA approved drug CQ can attenuate hypoxia-induced thrombosis through inhibition of autophagy and can be explored as an effective therapeutic alternative. HighlightsO_LIExposure of ex-vivo cultured platelets to physiologically relevant hypoxic condition (O2 concentration) can induce autophagy causing them to get activated and eventually aggregate. C_LIO_LIFDA approved drug chloroquine (CQ) is able to inhibit autophagy in anucleate cellular fragments, platelets, similar to nucleated cells and curb platelet functioning under hypoxic condition in both ex-vivo and in-vivo model. C_LIO_LIFlow restriction model mimicking deep vein thrombosis, emphasizes on the effect of CQ on impeding thrombus formation when used at a non-toxic dosage. C_LI

physiology↗

bioRxiv2024

Prediction of Ca2+ binding site in proteins with a fast and accurate method based on statistical mechanics and analysis of crystal structures

Predicting the precise locations of metal binding sites within metalloproteins is a crucial challenge in biophysics. A fast, accurate, and interpretable computational prediction method can complement the experimental studies. With this endeavor, In the current work, we have developed a method to predict the location of Ca2+ ions in calcium-binding proteins using a physics-based method with an all-atom description of the proteins, which is substantially faster than the molecular dynamics simulation-based methods with accuracy as good as data-driven approaches. Our methodology uses the three-dimensional reference interaction site model (3D-RISM), a statistical mechanical theory, to calculate Ca2+ ion density around protein structures, and the locations of the Ca2+ ions are obtained from the density. We have taken previously used datasets to assess the efficacy of our method as compared to previous works. Our accuracy is found to be 88%, comparable with the FEATURE program, one of the well-known data-driven methods. Moreover, our method being physical, the reasons for failures can be ascertained in most cases. We have thoroughly examined the failed cases using different structural and crystallographic measures, such as B-factor, R-factor, electron density map, and geometry at the binding site. It has been found that X-ray structures have issues in many of the failed cases, such as geometric irregularities and dubious assignment of ion positions. Our algorithm, along with the checks for structural accuracy, is a major step in predicting calcium ion positions in metalloproteins.

biophysics↗

bioRxiv2022

Anti-Biofilm Potential of Nanonized Eugenol against Pseudomonas aeruginosa

This study dealt with nanonization of eugenol, a major phytochemical present in basil leaf, which has pharmacological potential as an anti-bacterial agent. Eugenol nanoparticle (ENP) was synthesized by simple ultrasonic cavitation method through emulsification of hydrophobic eugenol into hydrophilic gelatin. Thus, the nanonization process made the water-insoluble eugenol to water-soluble nano-eugenol, making the nano-form bioavailable. The average size of the ENPs was 20-30 nm. Entrapment efficiency of eugenol within gelatin cap was about 80% of the eugenol, that was used as precursor in the nanonization reaction. In vitro release of eugenol from gelatin cap was slow and sustained over a period of five days. The ENP had higher anti-biofilm potency than eugenol for both formation and eradication of biofilm, formed by clinically relevant pathogen Pseudomonas aeruginosa. Minimal biofilm inhibitory concentration and minimal biofilm eradication concentration of ENPs were 2.0 and 4.0 mM respectively. In addition, the measurement of P. aeruginosa biofilm biomass, biofilm pellicle formation, biofilm thickness, amount of biofilm-forming extra-polymeric substance, cell surface hydrophobicity, cell swarming and twitching efficiencies, cellular morphology and biofilm formation in catheter demonstrated that the anti-biofilm efficacy of nano-eugenol was 30-40% higher than that of bulk eugenol. Thus, ENP can be used as a potential drug against pneumonia, a chronic infection in lung caused by P. aeruginosa, which is difficult to treat with antibiotics, due to natural intrinsic resistance of biofilm-formed cells to most antibiotics. The overall actions of ENP have been presented in the figure 1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/521144v1_fig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@12445e2org.highwire.dtl.DTLVardef@6813aforg.highwire.dtl.DTLVardef@e4779borg.highwire.dtl.DTLVardef@1688a9a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO Graphical abstract showing overall anti-biofilm action of ENP that is capable to inhibit each and every step of biofilm formation. ENP also carries the potential to destroy EPS and kill the biofilm residing cells by membrane disruption. Bacterial colonization has been found to be prevented significantly on ENP-coated catheter. C_FIG HighlightsO_LINano-formulation of eugenol, an important phytochemical, by ultrasonic cavitation method, which was simple, time-saving, low-cost and eco-friendly. C_LIO_LINanonization made water-insoluble eugenol into water-soluble form with enhanced therapeutic efficacy. C_LIO_LIThe eugenol nanoparticle (ENP) could inhibit formation of biofilm as well as facilitate eradication of pre-formed biofilm of P. aeruginosa. C_LIO_LIBiofilm formation was found to be prevented significantly on ENP-coated catheter. C_LIO_LINano-eugenol may be used as a potential drug against bacterial diseases, caused by pseudomonal biofilm, which are difficult to treat by antibiotics. C_LIO_LINano-formulated eugenol may also be used as an effective anti-fouling agent for biomedical devices like contact lens, pace-maker, materials for organ transplantation etc. to prevent bacterial colonization. C_LI

microbiology↗

bioRxiv2022

A strategy to optimize the peptide-based inhibitors against different mutants of the spike protein of SARS-CoV-2

SARS-CoV-2 virus has caused high-priority health concerns at a global level. Vaccines have stalled the proliferation of viruses to some extent. Yet, the emergence of newer, potentially more infectious, and dangerous mutants such as delta and omicron are among the major challenges in finding a more permanent solution for this pandemic. The effectiveness of antivirals Molnupiravir and Paxlovid, authorized for emergency use by the FDA, are yet to be assessed at larger populations. Patients with a high risk of disease progression or hospitalization have received treatment with a combination of antibodies (antibody-cocktail). Most of the mutations leading to the new lineage of SARS-CoV-2 are found in the spike protein of this virus that plays a key role in facilitating host entry. The current study has investigated how to modify a promising peptide-based inhibitor of spike protein, LCB3, against common mutations in the target protein so that it retains its efficacy against the spike protein. LCB3 being a prototype for protein-based inhibitors is an ideal testing system to learn about protein-based inhibitors. Two common mutations N501Y and K417N are considered in this work. Using a structure-based approach that considers free energy decomposition of residues, distance, and the interactions between amino acids, we propose the substitutions of amino acid residues of LCB3 inhibitors. Our binding free energy calculations suggest a possible improvement in the binding affinity of existing inhibitor LCB3 to the mutant forms of the S-protein using simple substitutions at specific positions of the inhibitor. This approach, being general, can be used in different inhibitors and other mutations and help in fighting against SARS-CoV-2.

biophysics↗