Presence Of Different Ligands Research Articles

Synthesized and characterization of Ce2(MoO4)2(Mo2O7)/MoO3 nanocomposites with Schiff-base ligand assistance as possible electrode materials for electrochemical energy storage

Transition metal oxides, which can deliver a cooperative impact through electrochemical processes, are now being investigated extensively as potential electrode materials for next-generation storage devices. Ce2(MoO4)2(Mo2O7)/MoO3 composites were created using hydrothermal technique aided by Schiff-base ligands in the low temperature and short time. Synthesis of composites in the presence of different ligands led to design different morphologies such as particles, plates, flower-shaped and spheres. The produced sample functions as an active component of the electrochemical energy storing arrangement, which is included of a KOH electrolyte and a three-electrode cell. Morphology affects both the durability of the compounds in the designed working electrode and the kinetics of the electrochemical process. Although, capacitance of Ce2(MoO4)2(Mo2O7)/MoO3 composites was considered at different morphologies for determining optimal performance. Results show ideal sample prepared in the presence of H2acacmda with microsphere morphology and surface area of 12.83 m2g−1 which demonstrates maximum capacity of 246 mAhg−1.

MultiModRLBP: A Deep Learning Approach for Multi-Modal RNA-Small Molecule Ligand Binding Sites Prediction.

This study aims to tackle the intricate challenge of predicting RNA-small molecule binding sites to explore the potential value in the field of RNA drug targets. To address this challenge, we propose the MultiModRLBP method, which integrates multi-modal features using deep learning algorithms. These features include 3D structural properties at the nucleotide base level of the RNA molecule, relational graphs based on overall RNA structure, and rich RNA semantic information. In our investigation, we gathered 851 interactions between RNA and small molecule ligand from the RNAglib dataset and RLBind training set. Unlike conventional training sets, this collection broadened its scope by including RNA complexes that have the same RNA sequence but change their respective binding sites due to structural differences or the presence of different ligands. This enhancement enables the MultiModRLBP model to more accurately capture subtle changes at the structural level, ultimately improving its ability to discern nuances among similar RNA conformations. Furthermore, we evaluated MultiModRLBP on two classic test sets, Test18 and Test3, highlighting its performance disparities on small molecules based on metal and non-metal ions. Additionally, we conducted a structural sensitivity analysis on specific complex categories, considering RNA instances with varying degrees of structural changes and whether they share the same ligands. The research results indicate that MultiModRLBP outperforms the current state-of-the-art methods on multiple classic test sets, particularly excelling in predicting binding sites for non-metal ions and instances where the binding sites are widely distributed along the sequence. MultiModRLBP also can be used as a potential tool when the RNA structure is perturbed or the RNA experimental tertiary structure is not available. Most importantly, MultiModRLBP exhibits the capability to distinguish binding characteristics of RNA that are structurally diverse yet exhibit sequence similarity. These advancements hold promise in reducing the costs associated with the development of RNA-targeted drugs.

Hot hole transfer at the plasmonic semiconductor/semiconductor interface.

Localized surface plasmon resonance (LSPR)-induced hot-carrier transfer provides an attractive alternative for light-harvesting using the full solar spectrum. This defect-mediated hot-carrier transfer is identical at the plasmonic semiconductor/semiconductor interface and can overcome the low efficiency of plasmonic energy conversion, thus boosting the efficiency of IR-light towards energy conversion. Here, using femtosecond transient absorption (TA) measurements, we directly observe the ultrafast non-radiative carrier dynamics of LSPR-driven hot holes created in CuS nanocrystals (NCs) and CuS/CdS hetero nanocrystals (HNCs). We demonstrate that in the CuS NCs, the relaxation dynamics follows multiple relaxation pathways. Two trap states are populated by the LSPR-induced hot holes in times (100-500 fs) that efficiently compete with the conventional LSPR mechanism (250 fs). The trapped hot holes intrinsically relax in 20-40 ps and then decay in 80 ns and 700 ns. In the CuS/CdS HNCs, once the CuS trap states have been populated by the LSPR-generated hot holes, the holes get transferred through plasmon induced transit hole transfer (PITCT) mechanism in 200-300 ps to the CdS acceptor phase and relax in 1-8 and 40-50 μs. The LSPR-recovery shows a weak excitation wavelength and fluence dependence, while the dynamics of the trap states remains largely unaffected. The direct observation of formation and decay processes of trap states and hole transfer from trap states provides important insight into controlling the LSPR-induced relaxation of degenerate semiconductors.

Analysis of Major Royal Jelly Proteins Stability and Ligand Binding by Differential Scanning Fluorimetry

Royal jelly (RJ) is produced by the hypopharingeal and mandibular glands of bees. Up to 90% of the RJ proteins are the major royal jelly proteins (MRJPs) that exhibit biological properties. Protein stability is of interest for biotechnology and can be explored by using differential scanning fluorimetry (DSF). We tested the stability by using a fluorescence dye (SYPRO® Orange,1:1000 dilution) in the presence of different ligands for MRJP1 and MRJP2 at 2 µM concentration. The fluorescence intensities (FI) were measured using the Real-Time PCR System and were fitted as a function of temperature according to the Boltzmann equation to determine the melting temperature (Tm) at which the amount of folded and unfolded protein is equal. In general, ligands stabilize the protein when binding to it and increase the Tm. Due to the increased starting fluorescence of the oligomeric MRJP1, the Tm values were only determined for monomeric MRJP1 and MRJP2. ATP, histamine, and thiamine were shown to determine an increase of Tm for the monomeric MRJP1 in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl. For the ligand ATP, the minimum limit of binding starts at 1.5 mM concentration, for the ligand Histamine it starts at 0.6 mM concentration, while for the ligand Thiamine it starts at 3 mM concentration.

Fe2+/Fe3+ based MOIFs + dye interaction study: Thermodynamic and kinetic analysis estimated through spectrophotometrically

Herein, the interaction study of organic dyes with newly reported metal organic ionic frameworks (MOIF) based on [Fe(CN)6]3-/[Fe(CN)6]4-/[Fe(CN)5NO]2- and cetylpyridinium cation, investigated through spectrophotometric method. Measurements were carried out at 303.15, 308.15 and 313.15 K where interaction found to be decreased with increase in temperatures. The 60–90% maximum interaction was noted for [Fe(CN)5NO]2- based MOIF with all chosen dyes at 303.15 K inferring the high interaction ability associated with dissymmetry of this MOIF due to presence of different ligands. The oxidation state and hydrophilicophobic domains of MOIFs have been found accountable for the stronger interaction where Fe2+ with high hydrophobicity was found more interacting as compared to Fe3+ with less hydrophobicity. The activation parameters ΔG0, ΔH0 have been found greater for MOIFs and methylene blue dye systems due to stronger interaction of their hydrophobic domains. The ΔS0 noted greater for [Fe(CN)5NO]2- based MOIF showing the impact of availability of interacting sites and distortion in symmetry. The isotherm analysis has inferred about the maximum uniform distribution of binding energy, favorability of interaction, interaction capacity and intensity, was associated with methylene blue and MOIFs systems. The positive slope (2.303/K) of a plot of log(Ao/At) vs t (time) inferred the first order kinetic of MOIF and chosen dyes.

Chemical and Physical Characterisation of Human Serum Albumin Nanocolloids: Kinetics, Strength and Specificity of Bonds with 99mTc and 68Ga.

Nanoparticles of Human Serum Albumin (NC) labelled with 99mTc are widely used in Nuclear Medicine and represent the gold-standard for the intraoperative detection of the sentinel lymph node in many kinds of cancer, mainly breast cancer and melanoma. A significant amount of radionuclides can be incorporated into the HSA particle, due to the multiple binding sites, and HSA-based nanocolloid catabolism is a fast and easy process that results in innocuous degradation products. NCs labelled with different isotopes represent an interesting radiopharmaceutical for extending diagnostic accuracy and surgical outcome, but the knowledge of the chemical bond between NCs and isotopes has not been fully elucidated, including information on its strength and specificity. The aim of this study is to investigate and compare the physicochemical characteristics of the bond between NCs and 99mTc and 68Ga isotopes. Commercial kits of HSA-based nanocolloid particles (NanoAlbumon®) were used. For this purpose, we have primarily studied the kinetic orders of NC radiolabelling. Langmuir isotherms and pH effect on radiolabelling were tested and the stability of the radiometal complex was verified through competition reactions carried out in presence of different ligands. The future goal of our research is the development of inexpensive and instant kits, easily labelled with a wide spectrum of diagnostic and therapeutic isotopes, thus facilitating the availability of versatile and multipurpose radiopharmaceuticals.

A novel method of water remediation of organic pollutants and industrial wastes by solution- route processed CZTS nanocrystals

CZTS (Cu2ZnSnS4), a P-type semiconductor with a direct bandgap (1.2–1.7eV), earth-abundant, non-toxic, and has a large absorption coefficient makes it extremely useful in optoelectronics and light-harvesting applications. In this work, CZTS is prepared by an ingenious, cost-effective colloidal route using the ‘hot-injection’ method with the usage of different ligands. The XRD and Raman spectroscopy shows the single-phase highly crystalline CZTS nanoparticles with kesterite structure. The TEM results show that the size of CZTS nanoparticles is about 2–5 nm and monodispersity is confirmed by DLS (Dynamic Light Scattering). FTIR confirms the presence of different ligands used in CZTS preparation. The Uv–vis absorption shows the direct bandgap of 1.5–1.7eV. The contact angle study shows the hydrophobic nature of as-synthesized CZTS nanoparticles which were further ligand exchanged with l-cysteine hydrochloride to make it hydrophilic to study the photocatalytic degradation activity of organic pollutants and industrial waste in the water. The photocatalysis experiments were performed under two conditions: (i) under bare sunlight (Intensity ∼ 900 W/m2) (ii) focussing the sample under the sunlight via converging lens (1800 W/m2). The photocatalytic efficiencies were then compared and the best photocatalytic efficiency achieved under sunlight was 98.4% for organic pollutants and 75% for industrial waste via converging lens while the corresponding efficiencies with bare sunlight were 98.1% and 73% respectively. To the best of the author’s knowledge, a rapid and highly efficient photocatalysis of CZTS NPs employing a converging lens for water-remediation without the usage of noble & transition-metals has been reported for the first time.

Identification of high-affinity inhibitors of SARS-CoV-2 main protease: Towards the development of effective COVID-19 therapy

Coronavirus disease 2019 (COVID-19) is an infectious disease, caused by a newly emerged highly pathogenic virus called novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Targeting the main protease (Mpro, 3CLpro) of SARS-CoV-2 is an appealing approach for drug development because this enzyme plays a significant role in the viral replication and transcription. The available crystal structures of SARS-CoV-2 Mpro determined in the presence of different ligands and inhibitor-like compounds provide a platform for the quick development of selective inhibitors of SARS-CoV-2 Mpro. In this study, we utilized the structural information of co-crystallized SARS-CoV-2 Mpro for the structure-guided drug discovery of high-affinity inhibitors from the PubChem database. The screened compounds were selected on the basis of their physicochemical properties, drug-likeliness, and strength of affinity to the SARS-CoV-2 Mpro. Finally, we have identified 6-Deaminosinefungin (PubChem ID: 10428963) and UNII-O9H5KY11SV (PubChem ID: 71481120) as potential inhibitors of SARS-CoV-2 Mpro which may be further exploited in drug development to address SARS-CoV-2 pathogenesis. Both compounds are structural analogs of known antivirals, having considerable protease inhibitory potential with improved pharmacological properties. All-atom molecular dynamics simulations suggested SARS-CoV-2 Mpro in complex with these compounds is stable during the simulation period with minimal structural changes. This work provides enough evidence for further implementation of the identified compounds in the development of effective therapeutics of COVID-19.

Fibril growth captured by electrical properties of amyloid-β and human islet amyloid polypeptide.

The aggregation of amyloid-β (Aβ) and human islet amyloid polypeptide (hIAPP) proteins have attracted considerable attention because of their involvement in protein misfolding diseases. These proteins have mostly been investigated using atomic force microscopy, transmission electron microscopy, and fluorescence microscopy to study the directional growth of fibrils both perpendicular to and along the fibril axis. Here, we demonstrate the real-time monitoring of the directional growth of fibrils in terms of activation energy of proton transfer using an impedance spectroscopy technique. The activation energy is used to quantify the sensitivity of proton conduction to the different stages of protein aggregation. The decrement (increment) in activation energy is related to the fibril growth along (perpendicular to) the fibril axis in intrinsic protein aggregation. The entire aggregation process shows different phases of the directional growth for Aβ and hIAPP, indicating different pathways for their aggregation. The activation energy for hIAPP is found to be smaller than the activation energy of Aβ during the aggregation process. The oscillatory behavior of the activation energy of hIAPP reflects a rapid change in the directional growth of the protofilaments of hIAPP. The results indicate higher aggregation propensity of Aβ than hIAPP. In the presence of resveratrol, hIAPP exhibits slower aggregation compared to Aβ. Methods of this study may in general be used to reveal the modulated aggregation pathway of proteins in the presence of different ligands.

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics-even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D2O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.

Reconstitution of the Jasmonate Signaling Pathway in Plant Protoplasts.

The phytohormone jasmonic acid (JA) plays an important role in various plant developmental processes and environmental adaptations. The JA signaling pathway has been well-elucidated in the reference plant Arabidopsis thaliana. It starts with the perception of the active JA derivative, jasmonoyl-isoleucine (JA-Ile), by the F-box protein COI1 which is part of the E3-ligase SCFCOI1. Binding of JA-Ile enables the interaction between COI1 and JAZ repressor proteins. Subsequent degradation of JAZ proteins leads to the activation of transcription factors like e.g., MYC2. Here we demonstrate that the pathway can be reconstituted in transiently transformed protoplasts. Analysis of the stability of a JAZ1-fLuc fusion protein as a function of COI1 transiently expressed in coi1 protoplasts allows structure function analysis of both JAZs and COI1. Using this system, we found that conserved cysteines in COI1 influence steady state COI1 protein levels. Using a luciferase reporter gene under the control of the JAZ1 promoter enable to address those features of JAZ1 that are required for MYC2 repression. Interestingly, the conserved TIFY-motif previously described to interact with NINJA to recruit the corepressor TOPLESS is not necessary for repression. This result is in favor of the alternative repression mode that proposes a direct competition between repressive JAZs and promotive MEDIATOR25 at MYC2. Finally, using protoplasts from the aos coi1 double mutant, which is deficient in JA synthesis and perception, we provide a system that has the potential to study the activity of different COI1 variants in the presence of different ligands.

Functional Roles of Metabolic Intermediates in Regulating the Human Mitochondrial NAD(P)+-Dependent Malic Enzyme

Human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME) has a dimer of dimers quaternary structure with two independent allosteric sites in each monomer. Here, we reveal the different effects of nucleotide ligands on the quaternary structure regulation and functional role of the human m-NAD(P)-ME exosite. In this study, size distribution analysis was utilized to investigate the monomer-dimer-tetramer equilibrium of m-NAD(P)-ME in the presence of different ligands, and the monomer-dimer (Kd,12) and dimer-tetramer (Kd,24) dissociation constants were determined with these ligands. With NAD+, the enzyme formed more tetramers, and its Kd,24 (0.06 µM) was 6-fold lower than the apoenzyme Kd,24 (0.34 µM). When ATP was present, the enzyme displayed more dimers, and its Kd,24 (2.74 µM) was 8-fold higher than the apoenzyme. Similar to the apoenzyme, the ADP-bound enzyme was present as a tetramer with a small amount of dimers and monomers. These results indicate that NAD+ promotes association of the dimeric enzyme into tetramers, whereas ATP stimulates dissociation of the tetrameric enzyme into dimers, and ADP has little effect on the tetrameric stability of the enzyme. A series of exosite mutants were created using site-directed mutagenesis. Size distribution analysis and kinetic studies of these mutants with NAD+ or ATP indicated that Arg197, Asn482 and Arg556 are essential for the ATP binding and ATP-induced dissociation of human m-NAD(P)-ME. In summary, the present results demonstrate that nucleotides perform discrete functions regulating the quaternary structure and catalysis of m-NAD(P)-ME. Such regulation by the binding of different nucleotides may be critically associated with the physiological concentrations of these ligands.

Insights into the Basal Activity and Activation Mechanism of the β1 Adrenergic Receptor Using Native Mass Spectrometry.

In the absence of orthosteric ligands, most G protein-coupled receptors (GPCRs) exist in an equilibrium of different conformational states. This equilibrium is shifted by an agonist towards the active state or by an inverse agonist towards the inactive state. The basal activity of the receptor, and its ability to activate intracellular signaling pathways, is defined by the probability that a fraction of the receptor adopts the active state in the absence of ligand. Despite breakthroughs in native MS of membrane proteins, GPCR-transducing complexes have not been studied by this approach until very recently. Here, we investigated different conformational states of the turkey β1 adrenergic receptor (tβ1AR) in complex with two transducing partners: a G protein mimicking nanobody, Nb80, and an engineered truncated Gs protein (miniGs), in the presence of the full agonist isoprenaline by native MS. Interestingly, complex formation with both transducing partners was also observed in the absence of agonist, and allowed us to quantify basal activity of tβ1AR. We followed the stepwise disassembly of the transducing complexes by increasing the concentration of the inverse agonist S32212 in the presence of a constant concentration of isoprenaline. This allowed us to determine the relative binding affinity of S32212 in comparison to isoprenaline by native MS. Our approach provides a fast and sensitive way to detect complexes, study their stability in the presence of different ligands, and determine relative ligand affinities. Native mass spectrometry thus has the potential to become a useful tool to screen for orthosteric and allosteric GPCR drugs. Graphical Abstract.

Quantum Dot Size Effect on the Frontier Molecular Orbital Energies in the Presence of Different Aquatic Environmental Ligands

One of the challenging tasks of the century is to clean up the contaminants of the environment by ecofriendly, sustainable and economically adoptable technologies. The application of quantum dots (QDs) is growing rapidly in to the field of nanotechnology. The electronic properties such as the energy of the highest occupied molecular orbital (EHOMO), the lowest unoccupied molecular orbital (ELUMO), the energy gap ΔE are correlated to the size of quantum dots (QDs) such as CdSe in the presence of different ligands. Linear regression equations were developed through statistical analysis using SPSS. In terms of correlation coefficient R2, both the size and nature of organic ligand capping affect the EHOMO, ELUMO and ΔE levels of QDs. Among all QDs, CdSe has the strongest correlation between its frontier molecular orbital energy levels with the QDs size. The R2 value is greater than 0.63 for a regression equation with a coefficient value of −0.17 and a constant of 2.9. This result implies that a unit decrease of size in the range lower than 7 nm would increase the band gap ΔE by 17%. Potential environmental applications of QDs are presented.

Polymer inclusion membrane to access Zn speciation: Comparison with root uptake

Metal speciation studies can be performed with a new technique based on a functionalized membrane. The estimation of not only the total amount of metal, but also the metal available to living organisms is very important. In this context, we have investigated the use of a polymer inclusion membrane (PIM) in a new tool for the determination of free metal ion concentration. In order to check the usefulness of PIM devices in metal speciation studies and metal availability to potato plants (Solanum tuberosum), Zn has been chosen as a case study. The PIM designed for Zn transport uses polyvinyl chloride (PVC) as polymer and di-(2-ethylhexyl) phosphoric acid (D2EHPA) as carrier, with 0.01M nitric acid in the receiving solution. The stability of the PIM has been demonstrated and good linearity of PIM-device fluxes (JPIM) with free metal concentration was observed for total metal concentrations ranging from 3μM up to 70μM. The presence of different ligands, such as ethylenediaminetetraacetic acid (EDTA), humic acid (HA) and citrate, greatly influences the measured JPIM because the formation of metal complexes in the donor phase decreases the free Zn concentration in the sample. Good correlation has been found when comparing PIM fluxes and metal accumulation in potato plants roots in the presence of EDTA. But, the root uptake did not change when adding citrate and HA to the hydroponic medium, so the uptake does not always follows the Free Ion Activity Model (FIAM). These ligands might induce physiological changes in the roots and enhance metal uptake.

The Met80Ala and Tyr67His/Met80Ala mutants of human cytochrome c shed light on the reciprocal role of Met80 and Tyr67 in regulating ligand access into the heme pocket

The spectroscopic and functional properties of the single Met80Ala and double Tyr67His/Met80Ala mutants of human cytochrome c have been investigated in their ferric and ferrous forms, and in the presence of different ligands, in order to clarify the reciprocal effect of these two residues in regulating the access of exogenous molecules into the heme pocket. In the ferric state, both mutants display an aquo high spin and a low spin species. The latter corresponds to an OH– ligand in Met80Ala but to a His in the double mutant. The existence of these two species is also reflected in the functional behavior of the mutants. The observation that (i) a significant peroxidase activity is present in the Met80Ala mutants, (ii) the substitution of the Tyr67 by His leads to only a slight increase of the peroxidase activity in the Tyr67His/Met80Ala double mutant with respect to wild type, while the Tyr67His mutant behaves as wild type, as previously reported, suggests that the peroxidase activity of cytochrome c is linked to an overall conformational change of the heme pocket and not only to the disappearance of the Fe-Met80 bond. Therefore, in human cytochrome c there is an interplay between the two residues at positions 67 and 80 that affects the conformation of the distal side of the heme pocket, and thus the sixth coordination of the heme.

Structural rearrangement of the intracellular domains during AMPA receptor activation

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact.

Revealing G-protein-coupled receptor oligomerization at the single-molecule level through a nanoscopic lens: methods, dynamics and biological function.

The introduction of super-resolution fluorescence microscopy has allowed the visualization of single proteins in their biological environment. Recently, these techniques have been applied to determine the organization of class A G-protein-coupled receptors (GPCRs), and to determine whether they exist as monomers, dimers and/or higher-order oligomers. On this subject, this review highlights recent evidence from photoactivated localization microscopy (PALM), which allows the visualization of single molecules in dense samples, and single-molecule tracking (SMT), which determines how GPCRs move and interact in living cells in the presence of different ligands. PALM has demonstrated that GPCR oligomerization depends on the receptor subtype, the cell type, the actin cytoskeleton, and other proteins. Conversely, SMT has revealed the transient dynamics of dimer formation, whereby receptors show a monomer-dimer equilibrium characterized by rapid association and dissociation. At steady state, depending on the subtype, approximately 30-50% of receptors are part of dimeric complexes. Notably, the existence of many GPCR dimers/oligomers is also supported by well-known techniques, such as resonance energy transfer methodologies, and by approaches that exploit fluorescence fluctuations, such as fluorescence correlation spectroscopy (FCS). Future research using single-molecule methods will deepen our knowledge related to the function and druggability of homo-oligomers and hetero-oligomers.

A user-friendly web portal for analyzing conformational changes in structures of Mycobacterium tuberculosis

Initiation of the Tuberculosis Structural Consortium has resulted in the expansion of the Mycobacterium tuberculosis (MTB) protein structural database. Currently, 969 experimentally solved structures are available for 354 MTB proteins. This includes multiple crystal structures for a given protein under different functional conditions, such as the presence of different ligands or mutations. In depth analysis of the multiple structures reveal that subtle differences exist in conformations of a given protein under varied conditions. Therefore, it is immensely important to understand the conformational differences between the multiple structures of a given protein in order to select the most suitable structure for molecular docking and structure-based drug designing. Here, we introduce a web portal ( http://bmi.icmr.org.in/mtbsd/torsion.php ) that we developed to provide comparative data on the ensemble of available structures of MTB proteins, such as Cα root means square deviation (RMSD), sequence identity, presence of mutations and torsion angles. Additionally, torsion angles were used to perform principal component analysis (PCA) to identify the conformational differences between the structures. Additionally, we present a few case studies to demonstrate this database. Graphical Abstract Conformational changes seen in the structures of the enoyl-ACP reductase protein encoded by the Mycobacterial gene inhA.

Elucidation of Ligand-Dependent Modulation of Disorder-Order Transitions in the Oncoprotein MDM2.

Numerous biomolecular interactions involve unstructured protein regions, but how to exploit such interactions to enhance the affinity of a lead molecule in the context of rational drug design remains uncertain. Here clarification was sought for cases where interactions of different ligands with the same disordered protein region yield qualitatively different results. Specifically, conformational ensembles for the disordered lid region of the N-terminal domain of the oncoprotein MDM2 in the presence of different ligands were computed by means of a novel combination of accelerated molecular dynamics, umbrella sampling, and variational free energy profile methodologies. The resulting conformational ensembles for MDM2, free and bound to p53 TAD (17-29) peptide identify lid states compatible with previous NMR measurements. Remarkably, the MDM2 lid region is shown to adopt distinct conformational states in the presence of different small-molecule ligands. Detailed analyses of small-molecule bound ensembles reveal that the ca. 25-fold affinity improvement of the piperidinone family of inhibitors for MDM2 constructs that include the full lid correlates with interactions between ligand hydrophobic groups and the C-terminal lid region that is already partially ordered in apo MDM2. By contrast, Nutlin or benzodiazepinedione inhibitors, that bind with similar affinity to full lid and lid-truncated MDM2 constructs, interact additionally through their solubilizing groups with N-terminal lid residues that are more disordered in apo MDM2.