Rapid Long-distance Migration of RPA on Single Stranded DNA Occurs Through Intersegmental Transfer Utilizing Multivalent Interactions.

Replication Protein A (RPA) is asingle strandedDNA(ssDNA)binding protein that coordinates diverse DNA metabolic processes including DNA replication, repair, and recombination. RPA is a heterotrimeric protein with six functional oligosaccharide/oligonucleotide (OB) domains and flexible linkers. Flexibility enables RPA to adopt multiple configurations andis thought to modulate its function. Here, usingsingle moleculeconfocal fluorescencemicroscopy combinedwith optical tweezers and coarse-grained molecular dynamics simulations, we investigated the diffusional migration of single RPA molecules on ssDNA undertension.The diffusioncoefficientDis the highest (20,000nucleotides2/s) at 3pNtension and in 100 mMKCl and markedly decreases whentensionor salt concentrationincreases. We attribute the tension effect to intersegmental transfer which is hindered by DNA stretching and the salt effect to an increase in binding site size and interaction energy of RPA-ssDNA. Our integrative study allowed us to estimate the size and frequency of intersegmental transfer events that occur through transient bridging of distant sites on DNA by multiple binding sites on RPA. Interestingly, deletion of RPA trimeric core still allowed significant ssDNA binding although the reduced contact area made RPA 15-fold more mobile. Finally, we characterized the effect of RPA crowding on RPA migration. These findings reveal how the high affinity RPA-ssDNA interactions are remodeled to yield access, a key step in several DNA metabolic processes.

EnCPdock: a web-interface for direct conjoint comparative analyses of complementarity and binding energetics in inter-protein associations.

CONTEXT: Protein-protein interaction (PPI) is a key component linked to virtually all cellular processes. Be it an enzyme catalysis ('classic type functions' of proteins) or a signal transduction ('non-classic'), proteins generally function involving stable or quasi-stable multi-protein associations. The physical basis for such associations is inherent in the combined effect of shape and electrostatic complementarities (Sc, EC) of the interacting protein partners at their interface, which provides indirect probabilistic estimates of the stability and affinity of the interaction. While Sc is a necessary criterion for inter-protein associations, EC can be favorable as well as disfavored (e.g., in transient interactions). Estimating equilibrium thermodynamic parameters (∆Gbinding, Kd) by experimental means is costly and time consuming, thereby opening windows for computational structural interventions. Attempts to empirically probe ∆Gbinding from coarse-grain structural descriptors (primarily, surface area based terms) have lately been overtaken by physics-based, knowledge-based and their hybrid approaches (MM/PBSA, FoldX, etc.) that directly compute ∆Gbinding without involving intermediate structural descriptors. METHODS: Here, we present EnCPdock ( https://www.scinetmol.in/EnCPdock/ ), a user-friendly web-interface for the direct conjoint comparative analyses of complementarity and binding energetics in proteins. EnCPdock returns an AI-predicted ∆Gbinding computed by combining complementarity (Sc, EC) and other high-level structural descriptors (input feature vectors), and renders a prediction accuracy comparable to the state-of-the-art. EnCPdock further locates a PPI complex in terms of its {Sc, EC} values (taken as an ordered pair) in the two-dimensional complementarity plot (CP). In addition, it also generates mobile molecular graphics of the interfacial atomic contact network for further analyses. EnCPdock also furnishes individual feature trends along with the relative probability estimates (Prfmax) of the obtained feature-scores with respect to the events of their highest observed frequencies. Together, these functionalities are of real practical use for structural tinkering and intervention as might be relevant in the design of targeted protein-interfaces. Combining all its features and applications, EnCPdock presents a unique online tool that should be beneficial to structural biologists and researchers across related fraternities.

Can the jigsaw puzzle model of protein folding re-assemble a hydrophobic core?

According to the "jigsaw puzzle" model of protein folding, the isomorphism between sequence and structure is substantially determined by the specific geometry of side-chain interactions, within the protein interior. In this work, we have attempted to predict the hydrophobic core of cyclophilin (LdCyp) from Leishmania donovani, utilizing a surface complementarity function, which selects for high goodness of fit between hydrophobic side-chain surfaces, rather in the manner of assembling a three-dimensional jigsaw puzzle. The computational core prediction method implemented here has been tried on two distinct scenarios, on the LdCyp polypeptide chain with native non-core residues and all core residues initially set to alanine, on a poly-glycine polypeptide chain. Molecular dynamics simulations appeared to indicate partial destabilization of the two designed sequences. However, experimental characterization of the designed sequences by circular dichroism (CD) spectroscopy and denaturant (GdmCl) induced unfolding, demonstrated disordered proteins. Stepwise reconstruction of the designed cores by cumulative sequential mutations identified the specific mutation (M122L) as primarily responsible for fold collapse and all design objectives were achieved upon rectifying this mutation. In summary, the study demonstrates regions of the core to contain highly specific (jigsaw puzzle-like) interactions sensitive to any perturbations and a predictive algorithm to identify such regions. A mutation within the core has been identified which exercises an inordinate influence on the global fold, reminiscent of metamorphic proteins. In addition, the computational procedure could predict substantial regions of the core (given main-chain coordinates) without any reference to non-core residues.

Probing conformational transitions of PIN1 from L. major during chemical and thermal denaturation.

PIN1 proteins are a class of peptidyl prolyl cis-trans isomerases (PPIases), which have been implicated in numerous cellular functions like cell cycle progression, transcriptional control, signal transduction, promotion of oncogenesis and host-parasite interactions. In this work, the unfolding mechanism of a single domain PIN1 from Leishmania major (LmPIN1) has been characterized during thermal and denaturant-induced unfolding by differential scanning calorimetry (DSC), fluorescence and circular dichroism. Further, MD simulations have been performed to structurally probe the possible stages of its unfolding process. Both the fluorescence and CD data confirm classical two-state unfolding transitions for urea and GdnHCl. The thermal unfolding of LmPIN1, characterized by DSC, could optimally be fitted to a non two-state transition curve exhibiting two Tm's (53 °C and 57 °C) suggesting the possibility of an intermediate. Thermal unfolding of the modeled LmPIN1 by MD simulation shows that the unfolding process is initiated by increased fluctuations (dynamics) spanning residues 70-80, followed by perturbations in the sheet system and disjuncture of helix-sheet packing. Importantly, simulation and fluorescence quenching studies clearly suggest the possibility of the presence of residual structures of LmPIN1 even after complete denaturation.

pH-labile and photochemically cross-linkable polymer vesicles from coumarin based random copolymer for cancer therapy.

The stability of a drug payload inside a nanocarrier at physiological environment and the release of the said drug at specific tumor cells in a sustainable manner are the two most important factors that determine the efficiency of a smart targeted drug-delivery system. In this work, 2-hydroxyethyl methacrylate and a coumarin-based methacrylate monomer containing ß-thiopropionate moiety were copolymerized via reversible addition-fragmentation chain transfer (RAFT) process, followed by characterization using NMR and GPC. The said copolymer self-assembled at physiological pH to form vesicular nano-aggregates which was confirmed using DLS, TEM and by fluorescence measurements. These vesicles were further stabilized by photochemical crosslinking via coumarin (2π + 2π) cycloaddition reaction. These cross-linked vesicles (CVs) exhibited a 38% reduction in premature drug leakage as compared to the uncross-linked vesicles (UCVs) at physiological pH. Additionally, a slow hydrolysis of the ß-thiopropionate moieties under mildly acidic conditions prevalent in tumor cells resulted in disassembly of the vesicles, thereby releasing the loaded drug in a sustainable manner. Studies related to in vitro toxicity, efficiency of cellular uptake and pH-responsive antineoplastic activity of doxorubicin (DOX) loaded in the cross-linked vesicles (CVs) toward cancer cell lines were undertaken. A significant reduction in IC50 was noticed for DOX-loaded CVs in comparison to free DOX toward MG63 cancer cell lines, making these vesicles as potent nanocarrier systems for cancer therapy.

Valorization of waste micro-algal biomass - collected from coke oven effluent treatment plant and evaluation of sorption potential for fluoride removal.

Fluoride contamination in groundwater is now becoming a global concern. In the present study, removal of fluoride using dry biomass (DBM) of a micro-algal consortium of Chlorococcum infusionum and Leptolyngbya foveolaurum, collected from a coke-oven effluent treatment plant, Durgapur, India, has been investigated. The large volume of algal bloom in the industrial effluent has created serious disposal issues and caused severe environmental concerns. A biosorption technique has been carried out to valorize the waste algae biomass into a potential adsorbent. Response Surface Methodology (RSM) is used to model and optimize fluoride removal. Maximum fluoride removal (72%) is obtained at pH 4, 5 mg/L initial fluoride concentration, 0.5 g/L adsorbent dose (AD), and 25 °C temperature during one-factor-at-a-time (OFAT) analysis. The optimum condition of removal as specified by RSM is - initial concentration of fluoride: 30 mg/L, pH: 4.5, AD: 3.5 g/L and temperature: 30 °C. FESEM-EDX, FTIR and BET isotherm studies are done to characterize raw and fluoride treated biomass. Lagergren first order kinetic model and Freundlich isotherm model, are found to analyze best kinetic and equilibrium data, respectively. Adsorption capacity of DBM has been found to be 34.36 mg/g. The kinetics of fluoride removal have been well described by COMSOL Multiphysics.

Arm-First Approach toward Cross-Linked Polymers with Hydrophobic Domains via Hypervalent Iodine-Mediated Click Chemistry.

In this work, synthesis of two cross-linked polymeric systems through isoxazoline ring formation using nitrile oxide-acrylate click chemistry has been described. In the first system, styrenic block copolymer with oxime-functionalized middle block was synthesized using S,S'-bis(α,α'-dimethyl-α″-acetic acid)trithiocarbonate as chain-transfer agent using reversible addition fragmentation chain-transfer technique. This block copolymer was further utilized to prepare core cross-linked star polymers by reacting with a four-arm acrylic cross-linker by employing environment-friendly, nontoxic PhI(OAc)2-mediated "click reaction" via the formation of isoxazoline ring. In the second system, two linear styrenic block copolymers, one containing oxime and another containing acrylate group, were reacted to form a cross-linked (CS) polymeric system. Formation of cross-linked polymers and isoxazoline ring was confirmed by Fourier transform infrared spectroscopy, gel permeation chromatography, NMR spectroscopy, and dynamic light scattering studies. Later, we also demonstrated that in aqueous medium these CS polymers produced polymeric nanoparticles (NPs), which can be used as potential carriers of hydrophobic drug molecules. The loading capacity of the hydrophobic domains has been investigated using coumarin dyes with varying hydrophobicity through steady-state and time-resolved spectroscopy studies. The polymeric NPs were also shown to successfully encapsulate a hydrophobic drug doxorubicin.

Evaluation of fluoride bioremediation and production of biomolecules by living cyanobacteria under fluoride stress condition.

Application of microalgae for defluoridation has gained interest in recent years. In the present study, bioremediation of fluoride using living cyanobacteria, Starria zimbabweensis, collected from wastewater of coke-oven effluent treatment plant, Durgapur, India, has been investigated. Initially, the cyanobacterial strain was grown in BG11 medium at 25°C, 45µmol/m2/s irradiation in 18h: 6h light:dark cycle in an algal incubator. Samples were withdrawn after 2 days interval and analyzed for its dry biomass and lipid content. Optimum inoculum size of 10% and age of 16th day were assessed based on maximum dry biomass (9.307 ± 0.01g/L) and lipid (244.05 ± 0.02mg/L) production. SEM-EDX and FTIR studies of both native and fluoride treated biomass were done to emphasize the changes. During kinetic study of defluoridation, initial fluoride concentration was varied in the range of 10-50mg/L. Maximum fluoride removal (66.6 ± 0.11%) and dry biomass (18.19 ± 0.12g/L) were obtained at 10mg/L fluoride concentration using 10% of 16th day's inoculum. Biomass and lipid content were found to increase 2 and 4 folds, respectively under fluoride stress condition. Furthermore, chlorophyll, carbohydrate and protein content of the biomass were also compared between control and fluoride contaminated conditions. Fatty Acid Methyl Ester (FAME) analysis was done using Gas Chromatography (GC) to compare the lipid profile of native and fluoride loaded strain.

Photoresponsive Block Copolymer Prodrug Nanoparticles as Delivery Vehicle for Single and Dual Anticancer Drugs.

In recent decades, drug delivery systems (DDSs) based on polymer nanoparticles have been explored due to their potential to deliver drugs with poor water solubility. Some of the limitations of nanoparticle-based DDSs can be overcome by developing an appropriate polymer prodrug. In this work, poly(NIPA)-b-poly(HMNPPA)-b-poly(PEGMA-stat-BA) was synthesized using reversible addition fragmentation chain transfer polymerization and Chlorambucil (Cbl), an anticancer drug, was conjugated to the copolymer via 3-(3-(hydroxymethyl)-4-nitrophenoxy)propyl acrylate (HMNPPA) units to prepare the prodrug. A few biotin acrylate (BA) units were also incorporated to bring potential targeting capability to the prodrug in the copolymer. This polymer prodrug formed spherical micellar nanoparticles in physiological conditions, which were characterized by dynamic light scattering and transmission electron microscopy measurements. The very low critical aggregation concentration (cac) (0.011 mg/mL) of the prodrug, as measured from Nile Red fluorescence, makes it stable against dilution. The polymer prodrug was shown to release Cbl on photoirradiation by soft UV (λ ≥ 365 nm) and laser (λ = 405 nm) light. The prodrug micellar nanoparticles were capable of encapsulating a second drug (doxorubicin, DOX) in their hydrophobic core. On photoirradiation with UV and laser light of the DOX-loaded nanoparticles, both Cbl and DOX were released. Light-induced breaking of photolabile ester bond resulted in the release of Cbl and caused disruption of the nanoparticles facilitating release of DOX. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay confirmed the nontoxicity of the polymers and effectiveness of the dual drug-loaded micellar nanoparticles toward cancer cells. Confocal microscopy results showed a better cellular internalization capability of the DOX-loaded nanoparticles in cancer cells, possibly due to the presence of cancer cell targeting biotin molecules in the polymer. This new photoresponsive potentially biocompatible and cancer cell-targeted polymer prodrug may be useful for delivery of single and/or multiple hydrophobic drugs.

A comparative study of removal of fluoride from contaminated water using shale collected from different coal mines in India.

Low-cost water defluoridation technique is one of the most important issues throughout the world. In the present study, shale, a coal mine waste, is employed as novel and low-cost adsorbent to abate fluoride from simulated solution. Shale samples were collected from Mahabir colliery (MBS) and Sonepur Bazari colliery (SBS) of Raniganj coalfield in West Bengal, India, and used to remove fluoride. To increase the adsorption efficiency, shale samples were heat activated at a higher temperature and samples obtained at 550 °C are denoted as heat-activated Mahabir colliery shale (HAMBS550) and heat-activated Sonepur Bazari colliery shale (HASBS550), respectively. To prove the fluoride adsorption onto different shale samples and ascertain its mechanism, natural shale samples, heat-activated shale samples, and their fluoride-loaded forms were characterized using scanning electron microscopy, energy dispersive X-ray analysis, X-ray diffraction study, and Fourier transform infrared spectroscopy. The effect of different parameters such as pH, adsorbent dose, size of particles, and initial concentration of fluoride was investigated during fluoride removal in a batch contactor. Lower pH shows better adsorption in batch study, but it is acidic in nature and not suitable for direct consumption. However, increase of pH of the solution from 3.2 to 6.8 and 7.2 during fluoride removal process with HAMBS550 and HASBS550, respectively, confirms the applicability of the treated water for domestic purposes. HAMBS550 and HASBS550 show maximum removal of 88.3 and 88.5 %, respectively, at initial fluoride concentration of 10 mg/L, pH 3, and adsorbent dose of 70 g/L.