Probable Conformations Research Articles

In silico modelling and molecular dynamics simulation studies of thiazolidine based PTP1B inhibitors

Protein tyrosine phosphatase 1B (PTP1B) has been identified as a negative regulator of insulin and leptin signalling pathway; hence, it can be considered as a new therapeutic target of intervention for the treatment of type 2 diabetes. Inhibition of this molecular target takes care of both diabetes and obesity, i.e. diabestiy. In order to get more information on identification and optimization of lead, pharmacophore modelling, atom-based 3D QSAR, docking and molecular dynamics studies were carried out on a set of ligands containing thiazolidine scaffold. A six-point pharmacophore model consisting of three hydrogen bond acceptor (A), one negative ionic (N) and two aromatic rings (R) with discrete geometries as pharmacophoric features were developed for a predictive 3D QSAR model. The probable binding conformation of the ligands within the active site was studied through molecular docking. The molecular interactions and the structural features responsible for PTP1B inhibition and selectivity were further supplemented by molecular dynamics simulation study for a time scale of 30 ns. The present investigation has identified some of the indispensible structural features of thiazolidine analogues which can further be explored to optimize PTP1B inhibitors.

Native chromosome conformation

Isolation of nuclei in an isotonic buffer retains chromosome loops and allows the probing of intrinsic loop conformation.

Grid-independent Descriptors (GRIND) Analysis and SAR Guided Molecular Docking Studies to Probe Selectivity Profiles of Inhibitors of Multidrug Resistance Transporters ABCB1 and ABCG2.

ATP-binding cassette (ABC) transporters, P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP/ABCG2) are major determinants of pharmacokinetic, safety and efficacy profiles of drugs thereby effluxing a broad range of endogenous substances across the plasma membrane. Overexpression of these transporters in various tumors is also implicated in the development of multidrug resistance (MDR) and thus, hampers the success of cancer chemotherapy. Modulators of these efflux transporters in combination with chemotherapeutics could be a promising concept to increase the effective intracellular concentration of anticancer drugs. However, broad and overlapped specificity for substrates and modulators of ABCB1 and ABCG2, merely induce toxicity and unwanted drug-drug interactions and thus, lead to late-stage failure of drugs. In present investigation, we aim to identify specific 3D structural requirements for selective inhibition of ABCB1 and ABCG2 transport function. GRID Independent Molecular Descriptor (GRIND) models of selective inhibitors of both transporters have been developed, using their most probable binding conformations obtained from molecular docking protocol. Our results demonstrated a dominant role of molecular shape and different H-bonding patterns in drug-ABCB1/ABCG2 selective interactions. Moreover, distinct distances of different pharmacophoric features from steric hot spots of the molecules provided a strong basis of selectivity for both transporters. Additionally, our results suggested the presence of two H-bond donors at a distance of 8.4-8.8 Å in selective modulators of ABCG2. Our findings concluded that molecular shape along with three dimensional pattern of Hbonding in MDR modulators play a critical role in determining the selectivity between the two targets.

Second-harmonic phase determination by real-time in situ interferometry.

Second Harmonic Generation (SHG) has emerged as a highly sensitive probe of protein conformation. SHG can also be used to determine the tilt angle of an SHG-active moiety bound to a surface-adsorbed protein through polarization-dependent measurements. However, due to the coherent nature of SHG, interference occurs between the SHG produced by the SHG-active moieties and background sources at a solid-liquid interface, obscuring the signal of interest. In order to separate the protein-specific signal from the background signal, the phase difference between these two different sources of SHG must be determined. Although the phase difference can be obtained through a conventional interferometric approach involving a phase-modulated SHG source external to the sample, it can be sensitive to drift and other instabilities. We present here a simple, convenient, and crucially, model-independent method to determine the phase difference for any system in which the intensity of SHG-active moieties can be varied. We demonstrate the approach with time-resolved measurements of an SHG-active labeled protein binding to a supported lipid bilayer surface using a total internal reflection (TIR) geometry. This approach requires no additional optics beyond what is required to measure SHG and is highly stable since the interferometry occurs in situ, within the sample over a nanometer length scale, rather than external to it. To validate our measurements and the general approach, we constructed a dual-beam, external SHG interferometer in a TIR geometry. We also validated our approach by applying the in situ method to previously published measurements of the phase difference, obtaining the same values without recourse to a specific adsorption model.

Toward Closing the Gap: Quantum Mechanical Calculations and Experimentally Measured Chemical Shifts of a Microcrystalline Lectin.

NMR chemical shifts are exquisitely sensitive probes for conformation and dynamics in molecules and supramolecular assemblies. Although isotropic chemical shifts are easily measured with high accuracy and precision in conventional NMR experiments, they remain challenging to calculate quantum mechanically, particularly in inherently dynamic biological systems. Using a model benchmark protein, the 133-residue agglutinin from Oscillatoria agardhii (OAA), which has been extensively characterized by us previously, we have explored the integration of X-ray crystallography, solution NMR, MAS NMR, and quantum mechanics/molecular mechanics (QM/MM) calculations for analysis of 13Cα and 15NH isotropic chemical shifts. The influence of local interactions, quaternary contacts, and dynamics on the accuracy of calculated chemical shifts is analyzed. Our approach is broadly applicable and expected to be beneficial in chemical shift analysis and chemical-shift-based structure refinement for proteins and protein assemblies.

Probing A-form DNA: A fluorescent aminosugar probe and dual recognition by anthraquinone-neomycin conjugates

Nucleic acids adopt a broad array of hydrogen-bonded structures that enable their diverse roles in the cell; even the familiar DNA double helix displays subtle architectural nuances that are sequence dependent. While there have been many approaches for recognition of B-form nucleic acids, A-form DNA recognition has lagged behind. Here, using a tight binding fluorescein-neomycin (F-neo) conjugate that can probe the electrostatic environment of A-form DNA major groove, we developed a fluorescent displacement assay to be used as a screen for DNA duplex-binding compounds. As opposed to intercalating dyes that can significantly perturb DNA structure, the groove binding F-neo allows the probing of native DNA conformation. In combination with the assay development and probing of DNA grooves, we also report the synthesis and binding of a series of neomycin-anthraquinone conjugates, two units with a known preference for binding GC rich DNA. The assay can be used to identify duplex DNA-binding compounds, as well as probe structural features of a target DNA duplex, and can easily be scaled up for high throughput screening of compound libraries.

Relaxation processes in an aromatic polyamide-imide and composites on its basis with hydrosilicate nanoparticles

Features of the relaxation behavior of an aromatic polyamide-imide and a composite with nanotubes of magnesium silicate with the structure of chrysotile have been studied by dynamic mechanical analysis, dielectric spectroscopy, and differential scanning calorimetry. Two secondary relaxation (β1 and β2) transitions have been found, the activation energies of these processes have been determined, and the solvent effect on the cooperativeness degree has been studied. Changes in the value of the apparent activation energy of the β1-process caused by the subsequent heating of polymeric and composite samples have been analyzed according to the Starkweather procedure. It has been shown that, as the solvent is released from the polyamide-imide film, the polymer exhibits increasing local mobility, which are predetermined by the structure of the molecular unit. Using the GAMESS software, we have assumed the most probable dimer conformations that correspond to two repeating units of the polyamide-imide molecule.

The European Young Chemists Award 2016

The future is in good hands! The European Young Chemist Award for 2016 was awarded at the 6th EuCheMS Chemistry Congress in Seville. Reflections on the participants and their chemistry from the Convener of the event, Professor Bruno Pignataro, are given in the Guest Editorial.

Multiscale modeling of poly(2-isopropyl-2-oxazoline) chains in aqueous solution

Poly(2-isopropyl-2-oxazoline) (PIPOX) is a thermo-responsive polymer exhibiting lower critical solution behaviour in water close to the human body temperature. PIPOX chains form crystalline nanoribbons in aqueous solutions above the critical temperature (Tc). Chain conformations in water prior to the crystallization is still much debated. In this study, a multiscale computational approach is used to investigate the conformations of PIPOX chains well-below and well-above Tc. Molecular dynamics (MD) simulations are performed to obtain single chain behaviour while dissipative particle dynamics (DPD) simulations are utilized to estimate the mesoscale morphologies of the polymer-solvent systems. Atomistic information is imposed on mesoscale beads by reverse-mapping, followed by MD simulations for relaxation. The most probable conformation obtained from the reverse-mapping is a helix with a pitch length of 15 monomers. The helical PIPOX chains are used to construct a triclinic unit cell that well-reproduce the experimental X-ray diffraction pattern for crystalline PIPOX nanoribbons formed in water above Tc. A pair of interacting helical PIPOX chains maintain their relative conformations at Tc, stabilized by hydrophobic interactions of the isopropyl side groups. The results are important in identifying a precursor helical conformation for PIPOX prior to crystallization in water above Tc. The new structure paves the way in using the interactions and phase transitions of thermo-responsive polymers for design purposes, in light of their potential in biomedical applications.

A molecular dynamics study of the effect of glycosidic linkage type in the hemicellulose backbone on the molecular chain flexibility.

The macromolecular conformation of the constituent polysaccharides in lignocellulosic biomass influences their supramolecular interactions, and therefore their function in plants and their performance in technical products. The flexibility of glycosidic linkages from the backbone of hemicelluloses was studied by evaluating the conformational freedom of the φ and ψ dihedral angles using molecular dynamic simulations, additionally selected molecules were correlated with experimental data by nuclear magnetic resonance spectroscopy. Three types of β-(1→4) glycosidic linkages involving the monosaccharides (Glcp, Xylp and Manp) present in the backbone of hemicelluloses were defined. Different di- and tetrasaccharides with combinations of such sugar monomers from hemicelluloses were simulated, and free energy maps of the φ - ψ space and hydrogen-bonding patterns were obtained. The glycosidic linkage between Glc-Glc or Glc-Man (C-type) was the stiffest with mainly one probable conformation; the linkage from Man-Man or Man-Glc (M-type) was similar but with an increased probability for an alternative conformation making it more flexible, and the linkage between two Xyl-units (X-type) was the most flexible with two almost equally populated conformations. Glycosidic linkages of the same type showed essentially the same conformational space in both disaccharides and in the central region of tetrasaccharides. Different probabilities of glycosidic linkage conformations in the backbone of hemicelluloses can be directly estimated from the free energy maps, which to a large degree affect the overall macromolecular conformations of these polymers. The information gained contributes to an increased understanding of the function of hemicelluloses both in the cell wall and in technical products.

Free volume distribution of branched poly(methyl methacrylates): Conformational probes study

In this work we studied the free volume distribution of the branched poly (methylmethacrylates) by the method of conformational probes. The freezing temperatures of the conformational transitions of the probes introduced into branched polymers were determined by FTIR spectra. The influence of covalently connected fullerene С60 on the freezing temperatures of conformational transitions was shown.

Synthesis, solid-state structures, and urease inhibition activities of new copper(II) complexes based on O,N,O-tridentate Schiff bases

Six new complexes of copper(II) coordinated with O,N,O-tridentate Schiff base dianions were synthesized and structurally characterized. The solid-state structures of 1–6 contain four-coordinate mononuclear copper(II) units with a slightly distorted square planar geometry. Complexes 1 and 4 derived from d-tyrosine have an infinite 1-D, right-handed helical chain, while 5 derived from l-tyrosine has an infinite 1-D, left-handed helical chain. Inhibitions of jack bean urease by 1–6 have been investigated, and potent inhibitory activities with IC50 range of 2.15 ± 0.11–32.12 ± 0.65 μM have been observed for these copper(II) complexes. A docking analysis using a DOCK program was conducted to position 4 into the jack bean urease active site to determine the probable binding conformation.

An explicit-solvent conformation search method using open software.

Computer modeling is a popular tool to identify the most-probable conformers of a molecule. Although the solvent can have a large effect on the stability of a conformation, many popular conformational search methods are only capable of describing molecules in the gas phase or with an implicit solvent model. We have developed a work-flow for performing a conformation search on explicitly-solvated molecules using open source software. This method uses replica exchange molecular dynamics (REMD) to sample the conformational states of the molecule efficiently. Cluster analysis is used to identify the most probable conformations from the simulated trajectory. This work-flow was tested on drug molecules α-amanitin and cabergoline to illustrate its capabilities and effectiveness. The preferred conformations of these molecules in gas phase, implicit solvent, and explicit solvent are significantly different.

FTIR spectroscopy of conformational probes introduced into fullerene-containing branched poly (methyl methacrylate)

In this paper we consider the effect of fullerene on the local molecular dynamics of the branched poly (methylmethacrylates). The temperatures of the secondary relaxation transitions were determined by FTIR spectra. It was shown that there is the secondary relaxation transition at 190 K in copolymer containing C60, while there are the relaxation transitions at 230 and 190 K in same copolymer without fullerene.

NMR study and computational assays of meclofenamic Na salt and β-cyclodextrin inclusion complex

Complexation in solution between meclofenamate sodium (MCF) and β-cyclodextrin was studied using one- and two-dimensional 1H NMR spectroscopy and molecular docking coupled with ab initio calculations. The large variation of chemical shifts from protons located inside the hydrophobic cavity of β-cyclodextrin, provided clear evidence of inclusion complexation. The stoichiometry of the inclusion complex was determined to be 1:1, using the method of continuous variation. To ascertain the solution geometry of the host–guest complex a ROESY experiment was carried out. The results suggested a preferential binding of the dichlorophenyl moiety of the guest molecule within the β-cyclodextrin cavity, conformation which also sustained by the theoretical computational investigations. The association constant of the inclusion complex was determined using 1H NMR titration method in solution followed by a non-linear least-square regression implemented in CONSTEQ, a software developed in our group. After a preliminary molecular docking investigation, the most probable conformation was subjected to ab initio calculations using SIESTA software package, to characterize more precisely the stabilization energy of the 1:1 inclusion complex.

DEER Sensitivity between Iron Centers and Nitroxides in Heme-Containing Proteins Improves Dramatically Using Broadband, High-Field EPR.

This work demonstrates the feasibility of making sensitive nanometer distance measurements between Fe(III) heme centers and nitroxide spin labels in proteins using the double electron–electron resonance (DEER) pulsed EPR technique at 94 GHz. Techniques to measure accurately long distances in many classes of heme proteins using DEER are currently strongly limited by sensitivity. In this paper we demonstrate sensitivity gains of more than 30 times compared with previous lower frequency (X-band) DEER measurements on both human neuroglobin and sperm whale myoglobin. This is achieved by taking advantage of recent instrumental advances, employing wideband excitation techniques based on composite pulses and exploiting more favorable relaxation properties of low-spin Fe(III) in high magnetic fields. This gain in sensitivity potentially allows the DEER technique to be routinely used as a sensitive probe of structure and conformation in the large number of heme and many other metalloproteins.

Differential Mobility Spectrometry-Hydrogen Deuterium Exchange (DMS-HDX) as a Probe of Protein Conformation in Solution.

Differential mobility spectrometry (DMS) is an ion mobility technique that has been adopted chiefly as a pre-filter for small- to medium-sized analytes (<1 000 Da). With the exception of a handful of studies that employ an analogue of DMS-field asymmetric waveform ion mobility spectroscopy (FAIMS)-the application of DMS to intact biomacromolecules remains largely unexplored. In this work, we employ DMS combined with gas-phase hydrogen deuterium exchange (DMS-HDX) to probe the gas-phase conformations generated from proteins that were initially folded, partially-folded, and unfolded in solution. Our findings indicate that proteins with distinct structural features in solution exhibit unique deuterium uptake profiles as function of their optimal transmission through the DMS. Ultimately we propose that DMS-HDX can, if properly implemented, provide rapid measurements of liquid-phase protein structural stability that could be of use in biopharmaceuticals development. Graphical Abstract ᅟ.

Fluorescent Protein Fret as a Probe of Protein Conformation in Vivo and in Vitro

While methods for the determination of protein structure in vitro are highly advanced, the question always remains as to whether the observations are representative of the molecule in its native environment. Fluorescence resonance energy transfer (FRET) is widely used to probe protein structure both in vitro and in vivo. Because of their ease of use, in vivo FRET measurements frequently rely on the cyan and yellow fluorescent proteins (i.e. CFP donor and YFP acceptor). The severe spectral overlap and significant direct excitation of the acceptor complicate extraction of true FRET efficiency. Furthermore, the large size and poor photophysical properties of fluorescent proteins (FPs) make them a less than ideal FRET pair. To benchmark the reliability of FP-FRET as a probe of protein conformation in vivo, we characterized a series of CFP-YFP tandems with varying linker lengths. Ensemble and single molecule fluorescence were used to measure FRET in vitro. We then measured FRET in live cells with total internal reflection fluorescence microscopy. Only by using a single filter cube with a ratiometric image splitter, were we able to get agreement between in vitro and in vivo measurements. However, measurements of the radius of hydration, using analytical ultracentrifugation and chromatography, revealed a much different size distribution than was suggested by the FRET dependence on the linker length between CFP and YFP. Our studies suggest that the conformation of our protein test set is the same in live cells and in solution. In vitro and in vivo measurements for each construct were in good agreement. Unfortunately, distances derived from FP-FRET appear to misrepresent the actual interchromophore distance.

Activation of Integrin: A Multiscale Computational Study

Despite the importance of integrin proteins in cell signal transduction and force generation, the mechanism of action is not understood at the atomic level. To date, no experimental method has been able to probe the structure of integrin proteins in their open, force-activated state. To gain a better understanding of the transformation of the inactive, closed, state to an active state of the integrin, we are employing advanced computational techniques that will allow us to more quickly sample the conformational change and probe the pathway of the transformation. To make connection with the experimental studies, we have first studied the effect of single and double amino acid substitutions to the wild type integrin. Our simulations reveal certain mutants which destabilize the closed state by increasing the kink angle of the extracellular region and distance between the transmembrane region of the alpha and beta subunits of the integrin. We have also developed a coarse-grained model based on our atomistic simulation data, and are using that model to probe the transition of integrin to the open state.

Lid Conformational Dynamics and Substrate Specificity of the Pili Constructing Sortase C Enzymes

Pili are elongated protein structures that enhance the adhesive abilities and virulence of bacteria. Therefore, understanding the pilin assembly process has important ramifications for the development of new antibacterial compounds. In streptococcus pnuenomae, pili are formed by three Sortase C (SrtC) enzymes. In contrast to the housekeeping SrtA class of proteins, these SrtC enzymes possess an N-terminal extension that is thought to constitute a flexible lid that modulates access to the active site during the pili construction process. In this work, we have used enhanced sampling and free energy molecular dynamics simulations to study the conformational dynamics and probe the lid opening mechanism of three wild-type SrtC enzymes (SrtC-1, SrtC-2, and SrtC-3), as well as for several experimentally studied SrtC-1 mutants. The salt bridge between the conserved aspartate residue in the lid region and arginine in the active site leads to a robust anchoring of the lid that is stable during long conventional molecular dynamics simulations of all the SrtC proteins. In contrast, the results suggest that the aforementioned mutations lead to a dynamic lid, in accordance with experimental findings. We then carried out umbrella sampling calculations for SrtC-1 and its mutants which showed that the wild-type had the highest barrier for lid opening. This indicates that opening of the active site likely require interactions with the sorting signal (SS) and possibly other regions of the pilin building block. Calculations on SrtC in complex with either the SS or SS containing pilin subunit were then implemented. The data shows that the conformation of the lid is altered in the presence of substrate as proposed above. We also explore the structural and energetics underpinnings of the exquisite substrate specificity observed experimentally in the SrtC class of enzymes.