Identification of allosteric hotspots regulating the ribosomal RNA binding by antibiotic resistance-conferring Erm methyltransferases

Antibiotic resistance via epigenetic methylation of ribosomal RNA is one of the most prevalent strategies adopted by multidrug resistant pathogens. The erythromycin-resistance methyltransferase (Erm) methylates rRNA at the conserved A2058 position and imparts resistance to macrolides such as erythromycin. However, the precise mechanism adopted by Erm methyltransferases for locating the target base within a complicated rRNA scaffold remains unclear. Here, we show that a conserved RNA architecture, including specific bulge sites, present more than 15 Å from the reaction center, is key to methylation at the pathogenic site. Using a set of RNA sequences site-specifically labeled by fluorescent nucleotide surrogates, we show that base flipping is a prerequisite for effective methylation and that distal bases assist in the recognition and flipping at the reaction center. The Erm–RNA complex model revealed that intrinsically flipped-out bases in the RNA serve as a putative anchor point for the Erm. Molecular dynamic simulation studies demonstrated the RNA undergoes a substantial change in conformation to facilitate an effective protein–rRNA handshake. This study highlights the importance of unique architectural features exploited by RNA to impart fidelity to RNA methyltransferases via enabling allosteric crosstalk. Moreover, the distal trigger sites identified here serve as attractive hotspots for the development of combination drug therapy aimed at reversing resistance

Recent advances in the use of laccase enzyme in deep eutectic solvents

Laccase enzymes have enormous applications in various fields including textile, bioremediation, water purification and biofuel industries. However, the utility of laccases is limited due to poor stability and activity under different industrial operating conditions. There has been extensive research on improving the activity of laccases by enzyme engineering and solvent engineering approaches. In solvent engineering approaches, laccase has been studied in non-aqueous solvents such as ionic liquids (ILs) and deep eutectic solvents (DESs). DESs are greener and more sustainable in nature compared to ILs. DESs have many advantages, for example, inexpensive, less toxic, and easy preparation methods. However, to the best of our knowledge, there are no comprehensive reviews focusing specifically on studies related to laccase in DESs. In this review, we discussed the laccase activity and stability in various DESs considering the existing literature. In addition, we also discussed about the correlation between laccase interaction with different types of DESs components and descriptors which can regulate the activity of laccase. Finally, we highlighted the need for future research along the lines of the laccase-DESs greener systems which can provide significant insights into the development of laccase-DESs systems leading to the green and sustainable methods for wide range of applications

Spectroscopic analysis to identify the binding site for Rifampicin on Bovine Serum Albumin

This article reports the interaction of rifampicin, one of the important antituberculosis drugs, with Bovine Serum Albumin (BSA). Herein, we have monitored the fluorescence properties of tryptophan (Trp) residue in BSA to understand the interactions between protein and rifampicin. Fluorescence intensity of BSA was quenched tremendously upon interacting with the drug. Using steady state and time-resolved spectroscopic tools the static and dynamic nature of quenching have been characterised. Time correlated single photon counting technique confirmed that out of two lifetime components ∼6.2 ns and ∼2.8 ns of BSA, the rifampicin has affected only the shorter lifetime component a lot that was assigned to Trp-213 residue. Hence, it was thought that the drug must have been located near to the amino acid residue. Molecular docking studies have revealed the structural information of drug-protein complex which supported the above conjecture, confirming the nearest tryptophan as Trp-213 to the complexing rifampicin molecule. © 2022 Elsevier B.V

Fluorescence Up-Conversion Studies of [2,2′-Bipyridyl]-3,3′-diol in Octyl-β‑d‑glucoside and Other Micellar Aggregates

In this present work, excited state double proton transfer dynamics (ESIDPT) of 2,2′-bipyridyl-3,3′-diol (BP­(OH)₂) molecules has been probed in a nontoxic, biocompatible sugar surfactant assembly, namely, octyl-β-d-glucoside (OBG) micelle with the help of steady state and fluorescence up-conversion techniques. Moreover, the ultrafast double proton transfer dynamics in conventional micelles (SDS, CTAB) and bile salts aggregates have been probed and compared. Interestingly, in all these supramolecular aggregates, the ESIDPT dynamics is found to follow sequential pathway; however, the time-scale of proton transfer dynamics varies from 11 to 30 ps. This difference in proton transfer time scale in different supramolecular aggregates has been explained in terms of accessibility of water molecules in the vicinity of probe

Prototropical and Photophysical Properties of Ellipticine inside the Nanocavities of Molecular Containers

Host–guest interactions between an anticancer drug, ellipticine (EPT), and molecular containers (cucurbitruils (CB<i>n</i>) and cyclodextrins (CD)) are investigated with the help of steady state and time-resolved fluorescence measurements. Our experimental results confirm the formation of 1:1 inclusion complexes with CB7 and CB8. The protonated form of EPT predominantly prevails in the inclusion complexes due to the stabilization achieved through ion–dipole interaction between host and positively charged drug. Drug does not form an inclusion complex with CB6, which is smaller in cavity size compared to either CB7 or CB8. In the case of cyclodextrins, α-CD does not form an inclusion complex, whereas β-CD forms a 1:1 inclusion complex with the protonated form of the drug, and the binding affinity of EPT with β-CD is less compared to CB7/CB8. Interestingly, in the case of γ-CD, drug exists in different forms depending on the concentration of the host. At lower concentration of γ-CD, 1:1 inclusion complex formation takes place and EPT exists in protonated form due to accessibility of water by the drug in the inclusion complex, whereas, at higher concentration, a 2:1 inclusion complex (γ-CD:EPT) is observed, in which EPT is completely buried inside the hydrophobic cavity of the capsule formed by two γ-CD molecules, and we believe the hydrophobic environment inside the capsule stabilizes the neutral form of the drug in the 2:1 inclusion complex. Deep insight into the molecular picture of these host–guest interactions has been provided by the docking studies followed by quantum chemical calculations

Excited State Proton Transfer Dynamics of Topotecan Inside Biomimicking Nanocavity

The excited state proton transfer (ESPT) dynamics of a potentially important anticancer drug, Topotecan (TPT), has been explored in aqueous reverse micelle (RM) using steady-state and time-resolved fluorescence measurements. Both the time-resolved emission spectrum and time-resolved area normalized emission spectrum infer the generation of excited state zwitterionic form of TPT from the excited state cationic form of TPT, as a result of ESPT process from the −OH group of TPT to the nearby water molecule. The ESPT dynamics were found to be severely retarded inside the nanocavities of RMs, yielding time constants of 250 ps to 1.0 ns, which is significantly slower than the dynamics obtained in bulk water (32 ps). The observed slow ESPT dynamics in RM compared to bulk water is mainly attributed to the sluggish hydrogen-bonded network dynamics of water molecules inside the nanocavity of RM and the screening of the sodium ions present at the interface

What Makes Thienoguanosine an Outstanding Fluorescent DNA Probe?

Thienoguanosine (thG) is an isomorphic guanosine (G) surrogate that almost perfectly mimics G in nucleic acids. To exploit its full potential and lay the foundation for future applications, 20 DNA duplexes, where the bases facing and neighboring thG were systematically varied, were thoroughly studied using fluorescence spectroscopy, molecular dynamics simulations, and mixed quantum mechanical/molecular mechanics calculations, yielding a comprehensive understanding of its photophysics in DNA. In matched duplexes, thG's hypochromism was larger for flanking G/C residues but its fluorescence quantum yield (QY) and lifetime values were almost independent of the flanking bases. This was attributed to high duplex stability, which maintains a steady orientation and distance between nucleobases, so that a similar charge transfer (CT) mechanism governs the photophysics of thG independently of its flanking nucleobases. thG can therefore replace any G residue in matched duplexes, while always maintaining similar photophysical features. In contrast, the local destabilization induced by a mismatch or an abasic site restores a strong dependence of thG's QY and lifetime values on its environmental context, depending on the CT route efficiency and solvent exposure of thG. Due to this exquisite sensitivity, thG appears ideal for monitoring local structural changes and single nucleotide polymorphism. Moreover, thG's dominant fluorescence lifetime in DNA is unusually long (9-29 ns), facilitating its selective measurement in complex media using a lifetime-based or a time-gated detection scheme. Taken together, our data highlight thG as an outstanding emissive substitute for G with good QY, long fluorescence lifetimes, and exquisite sensitivity to local structural changes

Urea Induced Unfolding Dynamics of Flavin Adenine Dinucleotide (FAD): Spectroscopic and Molecular Dynamics Simulation Studies from Femto-Second to Nanosecond Regime

Here, we investigate the effect of urea in the unfolding dynamics of flavin adenine dinucleotide (FAD), an important enzymatic cofactor, through steady state, time-resolved fluorescence spectroscopic and molecular dynamics (MD) simulation studies. Steady state results indicate the possibility of urea induced unfolding of FAD, inferred from increasing emission intensity of FAD with urea. The TCSPC and up-conversion results suggest that the stack–unstack dynamics of FAD severely gets affected in the presence of urea and leads to an increase in the unstack conformation population from 15% in pure water to 40% in 12 M urea. Molecular dynamics simulation was employed to understand the nature of the interaction between FAD and urea at the molecular level. Results depict that urea molecules replace many of the water molecules around adenine and isoalloxazine rings of FAD. However, the major driving force for the stability of this unstack conformations arises from the favorable stacking interaction of a significant fraction of the urea molecules with adenine and isoalloxazine rings of FAD, which overcomes the intramolecular stacking interaction between themselves observed in pure water

Dynamics of Methylated Cytosine Flipping by UHRF1

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Structural and Dynamical Impact of a Universal Fluorescent Nucleoside Analogue Inserted Into a DNA Duplex

Recently, a 3-hydroxychromone based nucleoside 3HCnt has been developed as a highly environment-sensitive nucleoside surrogate to investigate protein–DNA interactions. When it is incorporated in DNA, the probe is up to 50-fold brighter than 2-aminopurine, the reference fluorescent nucleoside. Although the insertion of 3HCnt in DNA was previously shown to not alter the overall DNA structure, the possibility of the probe inducing local effects cannot be ruled out. Hence, a systematic structural and dynamic study is required to unveil the 3HCnt’s limitations and to properly interpret the data obtained with this universal probe. Here, we investigated by NMR a 12-mer duplex, in which a central adenine was replaced by 3HCnt. The chemical shifts variations and nOe contacts revealed that the 3HCnt is well inserted in the DNA double helix with extensive stacking interactions with the neighbor base pairs. These observations are in excellent agreement with the steady-state and time-resolved fluorescence properties indicating that the 3HCnt fluorophore is protected from the solvent and does not exhibit rotational motion. The 3HCnt insertion in DNA is accompanied by the extrusion of the opposite nucleobase from the double helix. Molecular dynamics simulations using NMR-restraints demonstrated that 3HCnt fluorophore exhibits only translational dynamics. Taken together, our data showed an excellent intercalation of 3HCnt in the DNA double helix, which is accompanied by localized perturbations. This confirms 3HCnt as a highly promising tool for nucleic acid labeling and sensing