Structures Of Different Conformations Research Articles

Structure and conformational dynamics of cyclobutanecarboxaldehyde in the ground electronic state

The study of the structure and conformational dynamics of cyclobutanecarboxaldehyde molecule in the ground electronic state was carried out by various quantum chemical methods. The geometrical structure of different conformers was investigated, and the topography of the potential energy surfaces in the low-energy region was studied. Four low-frequency molecular vibrations such as symmetric ring-CHO-bending, ring puckering, antisymmetric ring-CHO-bending and torsion are described in detail. The application of the anharmonic approach based on the variational principle makes it possible to predict the energies of vibrational transitions and to estimate qualitatively the complexity of the forms of studied vibrations.

Describing inhibitor specificity for the amino acid transporter LAT1 from metainference simulations

The human L-type amino acid transporter 1 (LAT1; SLC7A5) is a membrane transporter of amino acids, thyroid hormones, and drugs such as the Parkinson’s disease drug levodopa (L-Dopa). LAT1 is found in the blood-brain barrier, testis, bone marrow, and placenta, and its dysregulation has been associated with various neurological diseases, such as autism and epilepsy, as well as cancer. In this study, we combine metainference molecular dynamics simulations, molecular docking, and experimental testing, to characterize LAT1-inhibitor interactions. We first conducted a series of molecular docking experiments to identify the most relevant interactions between LAT1’s substrate-binding site and ligands, including both inhibitors and substrates. We then performed metainference molecular dynamics simulations using cryoelectron microscopy structures in different conformations of LAT1 with the electron density map as a spatial restraint, to explore the inherent heterogeneity in the structures. We analyzed the LAT1 substrate-binding site to map important LAT1-ligand interactions as well as newly described druggable pockets. Finally, this analysis guided the discovery of previously unknown LAT1 ligands using virtual screening and cellular uptake experiments. Our results improve our understanding of LAT1-inhibitor recognition, providing a framework for rational design of future lead compounds targeting this key drug target.

A Focus on Unusual ECL2 Interactions Yields β2 -Adrenergic Receptor Antagonists with Unprecedented Scaffolds.

The binding pockets of aminergic G protein‐coupled receptors are often targeted by drugs and virtual screening campaigns. In order to find ligands with unprecedented scaffolds for one of the best‐investigated receptors of this subfamily, the β2‐adrenergic receptor, we conducted a docking‐based screen insisting that molecules would address previously untargeted residues in extracellular loop 2. We here report the discovery of ligands with a previously undescribed coumaran‐based scaffold. Furthermore, we provide an analysis of the added value that X‐ray structures in different conformations deliver for such docking screens.

The Xenobiotic Extrusion Mechanism of the MATE Transporter NorM_PS from Pseudomonas stutzeri

Multidrug resistance (MDR) in bacterial pathogens has become a severe threat to public health. Membrane transporters of the multidrug and toxic compound extrusion (MATE) family contribute critically to MDR, making them promising drug targets. Despite recent advances, structures in different conformations and the mechanistic details of their antiport cycle are still elusive. Here we studied NorM_PS, a representative MATE transporter from Pseudomonas stutzeri, using biochemical assays in combination with hydrogen/deuterium exchange-mass spectrometry. Our results confirm that the antiport is proton dependent and electroneutral with a stoichiometry of two protons per one doubly positively charged substrate. We investigated the conformational dynamics upon substrate binding, and our hydrogen/deuterium exchange-mass spectrometry analysis revealed an occlusion in the proposed binding site as well as a closure of the cytoplasmic cavity and formation of a periplasmic cavity. Together with the results of selected variants (D38N, D373N and Q376A), we propose a six-step rocker-switch model for NorM_PS, which also increases our understanding of related MATE transporters and may help to fight the burden of MDR.

Characterizing highly dynamic conformational states: The transcription bubble in RNAP-promoter open complex as an example.

Bio-macromolecules carry out complicated functions through structural changes. To understand their mechanism of action, the structure of each step has to be characterized. While classical structural biology techniques allow the characterization of a few "structural snapshots" along the enzymatic cycle (usually of stable conformations), they do not cover all (and often fast interconverting) structures in the ensemble, where each may play an important functional role. Recently, several groups have demonstrated that structures of different conformations in solution could be solved by measuring multiple distances between different pairs of residues using single-molecule Förster resonance energy transfer (smFRET) and using them as constrains for hybrid/integrative structural modeling. However, this approach is limited in cases where the conformational dynamics is faster than the technique's temporal resolution. In this study, we combine existing tools that elucidate sub-millisecond conformational dynamics together with hybrid/integrative structural modeling to study the conformational states of the transcription bubble in the bacterial RNA polymerase-promoter open complex (RPo). We measured microsecond alternating laser excitation-smFRET of differently labeled lacCONS promoter dsDNA constructs. We used a combination of burst variance analysis, photon-by-photon hidden Markov modeling, and the FRET-restrained positioning and screening approach to identify two conformational states for RPo. The experimentally derived distances of one conformational state match the known crystal structure of bacterial RPo. The experimentally derived distances of the other conformational state have characteristics of a scrunched RPo. These findings support the hypothesis that sub-millisecond dynamics in the transcription bubble are responsible for transcription start site selection.

Structure and function of an Antiporter

Membrane proteins are essential to transport molecules across biological membranes. This gateway task makes them important drug targets. About 60% of all approved drugs target membrane proteins. Transport of ions across membranes is essential for every cell to maintain physiological salt concentrations and to keep pH homeostasis. In the past years X-Ray structures of various secondary transporters have provided insight into the mechanisms of membrane transport. However, difficulties in expression, purification and crystallization of membrane proteins still restrict the number of available structures. For well-characterized secondary transporters such as LeuT (1), BetP (2) and Ca2+/H+-exchangers (3) crystal structures in different conformations and substrate binding states have been obtained. However, for many important classes of transport proteins, detailed structures are urgently needed to understand their mechanism of action and to guide drug development. We report crystal structures of 2 homologues of a new secondary transporter in different states, with or without substrate bound. These structures shed light on the transport mechanism of this important class of membrane transport proteins.

Studying Conformational Changes of Mhp1 using Unbiased All-Atom Molecular Simulations

Mhp1 is a secondary active transporter from nucleobase/cation symporter-1 (NCS1) family. We have developed the implicit membrane-explicit membrane (IM-EX) hybrid simulation method to study conformational changes in secondary active transporters (JMB. 404:506). This novel approach starts with IM simulations and certain confirmations from the implicit simulation are placed in an explicit membrane environment to get the final conformation. Studying conformational changes of Mhp1 is an important test to our IM-EX method. Three different conformations of Mhp1 are known, i.e., inward-open, outward-occluded and outward-open. For Mhp1, the implicit simulation is applied by using SGLD-fp method (JCP. 135: 204101). The main advantage of SGLD-fp method is to overcome the energy barriers in a reasonable amount of simulation time. Then, certain conformations that show structural change during the implicit simulation are simulated with MD in an explicit membrane. Crystal structures of different conformations were used as initial conformation for SGLD-fp simulations. Glu289 was protonated based on its pKa value. The 20-ns simulation for inward-open conformation showed opening in extracellular gate but no closure for intracellular gate. In case of outward-occluded conformation, the result showed opening of EC gate while IC gate is still closed. The final structure agrees with the outward-open conformation, which means SGLD-fp method can predict conformational changes in a short time. For the outward-open conformation, simulations with SGLD-fp maintained this conformation. Protonation of other residues, choosing different initial seeds and effect of sodium and substrate are currently being studied to see how these factors affect transition between states.

A novel method to examine individual RNA structures within a mixed population reveals insights into the HIV-1 RNA dimerization process

Different models have been proposed for the HIV-1 RNA monomer and dimer structures, often by studying mutants that disrupt the palindromic nature of the dimerisation initiation site (DIS) to stabilise the monomer. We have developed a new technique known as “ingel SHAPE (selective 2’OH acylation analysed by primer extension)”. Individual RNA structures within a mixed structural population are isolated by electrophoresis under native conditions, and secondary structural probing takes place within the gel. High-throughput analysis generates a secondary structural map of each RNA, making it possible to examine the structures of different conformers under native conditions, without the need to stabilise each structure by mutagenesis or by using non-physiological buffers. We have validated the technique using TAR RNA, and show it to be accurate and highly reproducible. We have then used it to show that under native conditions the unspliced HIV-1 RNA monomer and dimer have different acylation sensitivities across the packaging signal region, but that the DIS is unreactive in both monomer and dimer. These data imply that a structural switch takes place; further examination showed that our results fit most closely to a model where, in the monomer, the dimerisation initiation site interacts with the U5 region, and that the native dimer structure corresponds closely with previously proposed structures of the monomeric RNA. This new technique allows us to visualise the nucleotides involved in the RNA structural switch, and those that remain static.

DFT Conformational Study of Calix[6]arene: Hydrogen Bond

We have performed DFT calculations to investigate the conformational characteristics and hydrogen bonds of the calix[6]arene (1) and p-tert-butylcalix[6]arene (2). The structures of various conformers of 1 were optimized by using the B3LYP/6-31G(d,p) and /6-31+G(d,p) methods followed by single point calculation of MPW1PW91/ 6-31G(d,p). The relative stability of the conformers of 1 is in the following order: cone (pinched: most stable) > partial-cone > cone (winged) <TEX>$\sim$</TEX> 1,2-alternate <TEX>$\sim$</TEX> 1,2,3-alternate > 1,4-alternate > 1,3-alternate > 1,3,5-alternate. The structures of different conformers of 2 were optimized by using the B3LYP/6-31G(d,p) method followed by single point calculation of MPW1PW91/6-31G(d,p). The relative stability of the conformers of 2 is in the following order: cone (pinched) > 1,2-alternate > cone (winged) > 1,4-alternate <TEX>$\sim$</TEX> partial-cone > 1,2,3-alternate > 1,3,5alternate > 1,3-alternate. One of the important factors affecting the relative stabilities of the various conformers of the 1 and 2 is the number and strength of the intramolecular hydrogen bonds.

Molecular dynamics simulation exploration of cooperative migration mechanism of calcium ions in sarcoplasmic reticulum Ca2+‐ATPase

Calcium ATPase is a member of the P-type ATPase, and it pumps calcium ions from the cytoplasm into the reticulum against a concentration gradient. Several X-ray structures of different conformations have been solved in recent years, providing basis for elucidating the active transport mechanism of Ca2+ ions. In this work, molecular dynamics (MD) simulations were performed at atomic level to investigate the dynamical process of calcium ions moving from the outer mouth of the protein to their binding sites. Five initial locations of Ca2+ ions were considered, and the simulations lasted for 2 or 6 ns, respectively. Specific pathways leading to the binding sites and large structural rearrangements around binding sites caused by uptake of calcium ions were identified. A cooperative binding mechanism was observed from our simulation. Firstly, the first Ca2+ ion binds to site I, and then, the second Ca2+ ion approaches. The interactions between the second Ca2+ and the residues around site I disturb the binding state of site I and weaken its binding ability for the first bound Ca2+. Because of the electrostatic repulsion of the second Ca2+ and the electrostatic attraction of site II, the first bound Ca2+ shifts from site I to site II. Concertedly, the second Ca2+ binds to site I, forming a binding state with two Ca2+ ions, one at site I and the other at site II. Both of Glu908 and Asp800 coordinate with the two Ca2+ ions simultaneously during the concerted binding process, which is believed to be the hinge to achieve the concerted binding. In our simulations, four amino acid residues that serve as the channel to link the outer mouth and the binding sites during the binding process were recognized, namely Tyr837, Tyr763, Asn911, and Ser767. The analyses regarding the activity of the proteins via mutations of some key residues also supported our cooperative mechanism.

DFT Conformational Study of Calix[5]arene and Calix[4]arene: Hydrogen Bond

We have performed DFT calculations to investigate the conformational characteristics and hydrogen bonds of the p-tert-butylcalix[5]arene (1) and p-tert-butylcalix[4]arene (2). The structures of different conformers of 1 were optimized by using B3LYP/6-31+G(d,p) method. The relative stability of the various conformers of 1 is in the following order: cone (most stable) > 1,2-alternate > partial-cone > 1,3-alternate. The relative stability of four conformers of 2 is in the following order: cone (most stable) > partial-cone > 1,2-alternate > 1,3-alternate. The primary factor affecting the relative stabilities of the various conformers of the 1 and 2 are the number and strength of the intramolecular hydrogen bonds. The hydrogen-bond distances are discussed based on different calculation methods.

The Gastric H,K ATPase as a Drug Target

The recent progress in therapy if acid disease has relied heavily on the performance of drugs targeted against the H,K ATPase of the stomach and the H2 receptor antagonists. It has become apparent in the last decade that the proton pump is the target that has the likelihood of being the most sustainable area of therapeutic application in the regulation of acid suppression. The process of activation of acid secretion requires a change in location of the ATPase from cytoplasmic tubules into the microvilli of the secretory canaliculus of the parietal cell. Stimulation of the resting parietal cell, with involvement of F-actin and ezrin does not use significant numbers of SNARE proteins, because their message is depleted in the pure parietal cell transcriptome. The cell morphology and gene expression suggest a tubule fusion-eversion event. As the active H,K ATPase requires efflux of KCl for activity we have, using the transcriptome derived from 99% pure parietal cells and immunocytochemistry, provided evidence that the KCl pathway is mediated by a KCQ1/KCNE2 complex for supplying K and CLIC6 for supplying the accompanying Cl. The pump has been modeled on the basis of the structures of different conformations of the sr Ca ATPase related to the catalytic cycle. These models use the effects of site directed mutations and identification of the binding domain of the K competitive acid pump antagonists or the defined site of binding for the covalent class of proton pump inhibitors. The pump undergoes conformational changes associated with phosphorylation to allow the ion binding site to change exposure from cytoplasmic to luminal exposure. We have been able to postulate that the very low gastric pH is achieved by lysine 791 motion extruding the hydronium ion bound to carboxylates in the middle of the membrane domain. These models also allow description of the K entry to form the K liganded form of the enzyme and the reformation of the ion site inward conformation thus relating the catalytic cycle of the pump to conformational models. The mechanism of action of the proton pump inhibitor class of drug is discussed along with the cysteines covalently bound with these inhibitors. The review concludes with a discussion of the mechanism of action and binding regions of a possible new class of drug for acid control, the K competitive acid pump antagonists.

Distance measurements reveal a common topology of prokaryotic voltage-gated ion channels in the lipid bilayer

Voltage-dependent ion channels are fundamental to the physiology of excitable cells because they underlie the generation and propagation of the action potential and excitation-contraction coupling. To understand how ion channels work, it is important to determine their structures in different conformations in a membrane environment. The validity of the crystal structure for the prokaryotic K(+) channel, K(V)AP, has been questioned based on discrepancies with biophysical data from functional eukaryotic channels, underlining the need for independent structural data under native conditions. We investigated the structural organization of two prokaryotic voltage-gated channels, NaChBac and K(V)AP, in liposomes by using luminescence resonance energy transfer. We describe here a transmembrane packing representation of the voltage sensor and pore domains of the prokaryotic Na channel, NaChBac. We find that NaChBac and K(V)AP share a common arrangement in which the structures of the Na and K selective pores and voltage-sensor domains are conserved. The packing arrangement of the voltage-sensing region as determined by luminescence resonance energy transfer differs significantly from that of the K(V)AP crystal structure, but resembles that of the eukaryotic K(V)1.2 crystal structure. However, the voltage-sensor domain in prokaryotic channels is closer to the pore domain than in the K(V)1.2 structure. Our results indicate that prokaryotic and eukaryotic channels that share similar functional properties have similar helix arrangements, with differences arising likely from the later introduction of additional structural elements.

Ab initio Study of the Complexes of Trimethyl Ether of Monodeoxycalix[4]arene with Potassium Ion: Cation-π Interactions

In this study, we have performed ab initio computer simulations to investigate the conformational and complexation characteristics of the trimethyl ether of p-tert-butylmonodeoxycalix[4]arene (6) with a potassium ion. The structures of different conformers of 6 and their potassium complexes were optimized by using ab initio RHF/6-31G and B3LYP/6-31G(d,p) methods. The relative stability of the various conformers of the uncomplexed 6 is in following order: cone (most stable) > 1-partial-cone ~ 2i-partial-cone > 2-partial-cone ~ 1,3-alternate > 3i-partial-cone. However, the relative stability of the conformational complexes of 6 with <TEX>$K^+$</TEX> is in the following order: 2-partial cone ~ 1,3-alternate > cone > 3-partial cone > 1-partial cone (least stable). The highest binding strengths of 2-partial-cone and 1,3-alternate complexes originate from two strong cation-<TEX>$\pi$</TEX> interactions and two strong cation-oxygen interactions in the complex of 6+<TEX>$K^+$</TEX>. Due to the cation-<TEX>$\pi$</TEX> interactions, the calculated C-C bond distances in the arenes of the <TEX>$K^+$</TEX>-complexes are about 0.0048 <TEX>$\AA$</TEX> longer than the values of their isolated hosts.

Effect of a pentadentate Schiff base on the helical supramolecular structures of (μ-alkoxo)(μ-carboxylato)dicopper(II) complexes

Dicopper(II) complexes [Cu 2 L(O 2 CCH CHC 6 H 4 - p -OH)(MeOH)] · MeOH ( 1 · MeOH) and [Cu 2 L(O 2 CCH CHC 6 H 4 - p -OH)] · H 2 O ( 2 · H 2 O) having a carboxylate ligand with a p -hydroxycinnamyl moiety and the pentadentate Schiff base N , N ′-1,3-diyl-bis(acetylacetoneimine)propan-2-ol (H 3 L) in its trianionic form, are prepared and structurally characterized by X-ray crystallography. The antiferromagnetically coupled dicopper(II) complexes show hydrogen bonding interactions to form helical supramolecular structures of different conformations. Two new asymmetrically dibridged dicopper(II) complexes with a pentadentate Schiff base and p -hydroxycinnamate are prepared and structurally characterized by X-ray crystallography. The complexes have an asymmetrically dibridged (μ-alkoxo)(μ-carboxylato)dicopper(II) core with an alkoxo bridge from the Schiff base and the carboxylate showing a three-atom bridging mode. Variable-temperature magnetic studies show the complexes having an antiferromagnetically coupled spin system giving a singlet–triplet energy separation of −160 and −132 cm −1 for 1 and 2 , respectively. Complex 1 , with a shorter Cu–OR–Cu angle, displays greater antiferromagnetic spin coupling. Besides the Cu–OR–Cu angle, the role of the carboxylate ligand and the supramolecular structure in the spin coupling phenomena is observed. Complex 1 · MeOH shows the formation of intermolecular hydrogen bonds involving the axial methanol and the pentadentate Schiff base terminal oxygen atom. There is additional hydrogen bonding interactions involving the p -hydroxy group of the carboxylate, the lattice methanol and the terminal oxygen atom. The crystal structure of complex 2 · H 2 O displays the presence of a helical supramolecular structure due to hydrogen bonding interactions involving the pendant p -hydroxy group and the bridging oxygen atom of the carboxylate.

Conformational Analysis of Canonical 2-Deoxyribonucleotides. 2. Purine Nucleotides

The molecular structure of different conformers of isolated canonical purine 2′-deoxyribonu- cleotides 2-deoxyadenosine-5′-phosphate (pdA) and 2′-deoxyguanosine-5′-phosphate (pdG) was optimized using the B3LYP/6–31G(d) method. The results of the calculations reveal that the geometrical parameters and relative stability of the conformers significantly depend on the nature of the nucleobase, its orientation, the conformation of the furanose ring, the charge of the phosphate group and the character of the intramolecular hydrogen bonds. Analysis of the electron density distribution in purine nucleotides reveals the existence of a number of intramolecular hydrogen bonds. In general, the south conformer has a lower energy at the anti orientation of the base, and both conformers occur as the most stable for the syn orientation of the nucleobases. The results of the calculations reveal that the geometry and relative energy of the conformers of purine DNTs may be easily tuned by the charge of the phosphate group.

DFT and HF Studies of the Geometry, Electronic Structure, and Vibrational Spectra of 2-Nitrotetraphenylporphyrin and Zinc 2-Nitrotetraphenylporphyrin

The ground-state geometries and electronic structures of 2-nitrotetraphenylporphyrin (H2-2-NO2-TPP) and zinc 2-nitrotetraphenylporphyrin (Zn-2-NO2-TPP) with Cs and C1 symmetry have been determined from density functional theory, using the Becke−Lee−Yang−Parr composite exchange-correlation functional (B3-LYP) and ab initio RHF method and 6-31G(d) basis set. The optimized geometries are then compared with the crystallographic data of related compounds. The energy and electronic structures of different conformers are analyzed and compared with each other. The conformers with C1 symmetry are found to be more stable than that of Cs symmetry. The relative order of the highest occupied a2u and a1u orbitals determined by B3LYP (a2u > a1u) is reversed by RHF (a1u > a2u). The vibrational wavenumber, IR, and Raman intensities are also calculated at B3LYP/6-31G(d) optimized geometries. The calculated wavenumbers are scaled by a uniform scaling factor and compared with the experimental one. Most of the scaled modes are found to be in good agreement with the observed fundamentals. A single β-NO2 substitution slightly changes the geometries, the vibrational wavenumbers, and the frontier orbitals energy level.

The morph server: a standardized system for analyzing and visualizing macromolecular motions in a database framework.

The number of solved structures of macromolecules that have the same fold and thus exhibit some degree of conformational variability is rapidly increasing. It is consequently advantageous to develop a standardized terminology for describing this variability and automated systems for processing protein structures in different conformations. We have developed such a system as a 'front-end' server to our database of macromolecular motions. Our system attempts to describe a protein motion as a rigid-body rotation of a small 'core' relative to a larger one, using a set of hinges. The motion is placed in a standardized coordinate system so that all statistics between any two motions are directly comparable. We find that while this model can accommodate most protein motions, it cannot accommodate all; the degree to which a motion can be accommodated provides an aid in classifying it. Furthermore, we perform an adiabatic mapping (a restrained interpolation) between every two conformations. This gives some indication of the extent of the energetic barriers that need to be surmounted in the motion, and as a by-product results in a 'morph movie'. We make these movies available over the Web to aid in visualization. Many instances of conformational variability occur between proteins with somewhat different sequences. We can accommodate these differences in a rough fashion, generating an 'evolutionary morph'. Users have already submitted hundreds of examples of protein motions to our server, producing a comprehensive set of statistics. So far the statistics show that the median submitted motion has a rotation of approximately 10 degrees and a maximum Calpha displacement of 17 A. Almost all involve at least one large torsion angle change of >140 degrees. The server is accessible at http://bioinfo.mbb.yale. edu/MolMovDB

Partial heat capacity change — Fundamental characteristic of the process of thermal denaturation of biological macromolecules (proteins and nucleic acids)

We prove experimentally that for any biopolymers (proteins and nucleic acids), the process of thermal denaturation in aqueous medium is characterized by change of the heat capacity regardless a unique spatial structures of different conformations. Absolute values and sign of ΔC P= C p, denatured− C p, native, depends primarily on conformational peculiarities of macromolecules and concrete mechanisms of interaction of water molecules with biopolymers chains — interaction is very clearly found to be of critical importance for the stability of the spatial structure of any biopolymers in aqueous medium.

Serum carotenoids, vitamin E, and selenium as predictors of cancer death: John E. Connett, Ph.D.,∗ Marcus Kjelsberg, Ph.D.,† Lewis Kuller, Ph.D., D.Ph.,† and Gary Collins,† ∗Division of Biometry, University of Minnesota, Minneapolis, Minnesota 55414, and †Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Multidrug resistance (MDR) in bacterial pathogens has become a severe threat to public health. Membrane transporters of the multidrug and toxic compound extrusion (MATE) family contribute critically to MDR, making them promising drug targets. Despite recent advances, structures in different conformations and the mechanistic details of their antiport cycle are still elusive. Here we studied NorM_PS, a representative MATE transporter from Pseudomonas stutzeri, using biochemical assays in combination with hydrogen/deuterium exchange-mass spectrometry. Our results confirm that the antiport is proton dependent and electroneutral with a stoichiometry of two protons per one doubly positively charged substrate. We investigated the conformational dynamics upon substrate binding, and our hydrogen/deuterium exchange-mass spectrometry analysis revealed an occlusion in the proposed binding site as well as a closure of the cytoplasmic cavity and formation of a periplasmic cavity. Together with the results of selected variants (D38N, D373N and Q376A), we propose a six-step rocker-switch model for NorM_PS, which also increases our understanding of related MATE transporters and may help to fight the burden of MDR.