The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach

The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed.     

Phosphorylation analysis of G protein-coupled receptor by mass spectrometry: identification of a phosphorylation site in V2 vasopressin receptor

Phosphorylation plays vital roles in the regulation and function of the V2 vasopressin receptor (V2R), a G protein-coupled receptor (GPCR) that is responsible for maintaining water homeostasis in the kidney. Through a combination of immunoaffinity purification, immobilized metal affinity chromatography, and nanoflow liquid chromatography tandem mass spectrometry, we identified a novel phosphorylation site (Ser(255)) in the third intracellular loop of human V2R. We showed that the third intracellular loop could be phosphorylated in vitro by protein kinase A, but not by Akt kinase, although sequence motif analysis predicated otherwise. The analytical procedures and methodologies described in this study should be generally applicable for identifying the endogenous phosphorylation sites in other GPCRs, overcoming the limitations of conventional approaches such as sequence motif analysis and site-directed mutagenesis.     

Real-time monitoring of morphological changes in living cells by electronic cell sensor arrays: an approach to study G protein-coupled receptors

G protein-coupled receptors (GPCRs) constitute important targets for drug discovery against a wide range of ailments including cancer, inflammatory, and cardiovascular diseases. Efforts are underway to screen selective modulators of GPCRs and also to deorphanize GPCRs with unidentified natural ligands. Most GPCR-based cellular screens depend on labeling or recombinant expression of receptor or reporter proteins, which may not capture the true physiology or pharmacology of the GPCRs. In this paper, we describe a noninvasive and label-free assay for GPCRs that can be used with both engineered and nonengineered cell lines. The assay is based on using cell-electrode impedance to measure minute changes in cellular morphology as a result of ligand-dependent GPCR activation. We have used this technology to assay the functional activation of GPCRs coupled to different signaling pathways and have compared it to standard assays. We have used pharmacological modulators of GPCR signaling pathways to demonstrate the specificity of impedance-based measurements. Our data indicate that cell-electrode impedance measurements offer a convenient, sensitive, and quantitative method for assessing GPCR function. Moreover, the noninvasive nature of the readout offers the added advantage of performing multiple treatments in the same well to study events such as desensitization and receptor cross-talk.     

Systematic study on G‐protein couple receptor prototypes: did they really evolve from prokaryotic genes?

G‐protein couple receptor (GPCR) is one of the most striking examples of signalling proteins and it is only observed in eukaryotes. Based on various GPCR identification methods and classification systems, several evolutionary presumptions of different GPCR families have been reported. However, the prototype of GPCR still limits our knowledge. By investigating its structure and domain variance, the authors propose that GPCR might be evolved from prokaryotic world. The results given by the authors indicate that metabotropic glutamate receptor family would be the ancestor of GPCR. Phylogenetic analysis hints that one of metabotropic glutamate receptor GABA is possibly formed and evolved from the ancient chemical union of bacteriorhodopsin and periplasmic binding protein. The results obtained by the authors also unprecedentedly demonstrate that specific domains and identical structures are shown in each type of GPCR, which provides unique opportunities for future strategies on GPCR orphans’ prediction and classification.Inspec keywords: proteins, molecular biophysics, genetics, biochemistry, molecular configurations, bioinformatics, data miningOther keywords: systematic study, G‐protein couple receptor prototypes, prokaryotic genes, GPCR, signalling proteins, eukaryotes, metabotropic glutamate receptor family, phylogenetic analysis, GABA, bacteriorhodopsin, periplasmic binding protein, chemical union, GPCR orphans prediction, GPCR orphans classification     

Air-stable G protein-coupled receptor microarrays and ligand binding characteristics

This paper described novel strategies to achieve air-stable G protein-coupled receptor (GPCR) microarrays and the uses of the microarrays for ligand profiling. Specifically, GPCR cell membrane fragments were suspended in a buffered solution containing bovine serum albumin (BSA) and disaccharide sucrose or trehalose and used for fabricating GPCR microarrays. During the array fabrication and postfabrication processes, BSA molecules were found to effectively form packed layer(s) surrounding the GPCR membranes immobilized onto the predetermined printing area, thereby stabilizing the membrane microspots. The use of disaccharides was shown to protect the integrity and functionality of GPCR microarrays from the typical deterioration of the membranes when fabricated and stored under dry conditions. To utilize the ability of fluorescence technology for multichannel detection as well as to maximize the capability of GPCR microarrays for multiplexed binding assays, several fluorescently labeled ligands were synthesized and optimized for multiplexing binding assays. A schematic microarray of five GPCRs had been used as a model for characterizing the association and dissociation rate constants of labeled ligands binding to their respective receptors in the microarrays. Interestingly, distinct receptor-ligand interactions exhibited different dependence on the type of pH reagent as well as the species and concentration of cations used in a binding assay buffered solution. The potential mechanisms and implications for the uses of air-stable GPCR microarrays were discussed.     

Topological network alignment uncovers biological function and phylogeny

Sequence comparison and alignment has had an enormous impact on our understanding of evolution, biology and disease. Comparison and alignment of biological networks will probably have a similar impact. Existing network alignments use information external to the networks, such as sequence, because no good algorithm for purely topological alignment has yet been devised. In this paper, we present a novel algorithm based solely on network topology, that can be used to align any two networks. We apply it to biological networks to produce by far the most complete topological alignments of biological networks to date. We demonstrate that both species phylogeny and detailed biological function of individual proteins can be extracted from our alignments. Topology-based alignments have the potential to provide a completely new, independent source of phylogenetic information. Our alignment of the protein–protein interaction networks of two very different species—yeast and human—indicate that even distant species share a surprising amount of network topology, suggesting broad similarities in internal cellular wiring across all life on Earth.     

Surface plasmon resonance study of g protein/receptor coupling in a lipid bilayer-free system

Surface plasmon resonance (SPR) spectroscopy is a technique to study protein-protein interactions in real time; however, application of SPR spectroscopy for investigations of membrane receptors is difficult with respect to functional and uniform immobilization of receptors on a biosensor surface. In the current study, we developed a simple, direct, biosensor-based approach to monitor the molecular interactions between G protein transducin (Gt) and rhodopsin (Rho), a prototypical G protein-coupled receptor (GPCR). Detergent-solubilized dark-adapted Rho was captured onto a biosensor surface via lectin interaction, enabling site-directed immobilization of the receptor that made its cytoplasmic surface accessible to a coupling G protein. The system resembled the natural system with respect to receptor density, binding of Gt following flash or constant light application, fast GTP-dependent dissociation of Gt from Rho, regeneration of Rho, and dependence of Gt binding on light intensity and on concentration of Gt. The apparent KD of the Gt/Rho interaction was 13.6 nM. Our results validate the use of SPR spectroscopy as a tool to study G protein activation in GPCR systems and could be extended for application to other interaction partners of GPCRs.     

The influence of combined alignments on lateral acceleration on mountainous freeways: a driving simulator study

Combined horizontal and vertical alignments are frequently used in mountainous freeways in China; however, design guidelines that consider the safety impact of combined alignments are not currently available. Past field studies have provided some data on the relationship between road alignment and safety, but the effects of differing combined alignments on either lateral acceleration or safety have not systematically examined. The primary reason for this void in past research is that most of the prior studies used observational methods that did not permit control of the key variables. A controlled parametric study is needed that examines lateral acceleration as drivers adjust their speeds across a range of combined horizontal and vertical alignments. Such a study was conducted in Tongji University’s eight-degree-of-freedom driving simulator by replicating the full range of combined alignments used on a mountainous freeway in China. Multiple linear regression models were developed to estimate the effects of the combined alignments on lateral acceleration. Based on these models, domains were calculated to illustrate the results and to assist engineers to design safer mountainous freeways.     

Asymmetric Fullerene Nanosurfactant: Interface Engineering for Automatic Molecular Alignments

Since the molecular self‐assembly of nanomaterials is sensitive to their surface properties, the molecular packing structure on the surface is essential to build the desired chemical and physical properties of nanomaterials. Here, a new nanosurfactant is proposed for the automatic construction of macroscopic surface alignment layer for liquid crystal (LC) molecules. An asymmetric nanosurfactant (C₆₀NS) consisted of mesogenic cyanobiphenyl moieties with flexible alkyl chains and a [60]fullerene nanoatom is newly designed and precisely synthesized. The C₆₀NS directly introduced in the anisotropic LC medium is self‐assembled into the monolayered protrusions on the surface because of its amphiphilic nature originated by asymmetrically programmed structural motif of LC‐favoring moieties and LC‐repelling groups. The monolayered protrusions constructed by the phase‐separation and self‐assembly of asymmetric C₆₀NS nanosurfactant in the anisotropic LC media amplify and transfer the molecular orientational order from surface to bulk, and finally create the automatic vertical molecular alignment on the macroscopic length scale. The asymmetric C₆₀NS nanosurfactant and its self‐assembly described herein can offer the direct guideline of interface engineering for the automatic molecular alignments.     

Magnetism investigation of GaN monolayer doped with group VIII B transition metals

In order to enrich the potential of gallium nitride (GaN) monolayer in low-dimensional spintronic devices, we implement a theoretical study of the magnetic properties of group VIII B transition-metal (TM)-substituted GaN monolayer (ML). The results show that the group VIII B TM atom-substituted systems exhibit a distinct nonzero magnetic moment. The total magnetic moment of the Fe- and Ru-substituted systems is 5 μ_B, while that of Co- and Rh-substituted systems is 4 μ_B. Asymmetry of the degenerate and nondegenerate states, based on whether, and how, they are occupied, or not occupied by electrons in spin-up and spin-down bands decides the magnetic moment. The origin of magnetic moment is polarization of TM 3d or 4d electrons and N 2p electrons. Furthermore, co-doping of different TM atoms can increase the magnetic moment of GaN ML.     

Crystal growth and structure of L-methionine L-methioninium hydrogen maleate—a new NLO material

Abstract

A new organic nonlinear optical (NLO) crystal from the amino acid family, viz., L-methionine L-methioninium hydrogen maleate (LMMM), has been grown by slow evaporation method from aqueous solution. Bulk crystals were grown using submerged seed solution method. The structure was elucidated using the single crystal x-ray diffraction data. The compound crystallized in the space group P2₁ and the unit cell contains a protonated L-methioninium cation and a zwitterionic methionine residue plus a maleate anion. The backbone conformation angles Ψ¹ and Ψ² are in cis and trans configurations for both the methionine and methioninium residues, respectively. Amino and carboxyl groups of the methioninium and methionine residues are connected through N–H…O hydrogen bonds leading to a ring R₂²(10) motif.     

Crystal growth and structure of L-methionine L-methioninium hydrogen maleate—a new NLO material

A new organic nonlinear optical (NLO) crystal from the amino acid family, viz., L-methionine L-methioninium hydrogen maleate (LMMM), has been grown by slow evaporation method from aqueous solution. Bulk crystals were grown using submerged seed solution method. The structure was elucidated using the single crystal x-ray diffraction data. The compound crystallized in the space group P2₁ and the unit cell contains a protonated L-methioninium cation and a zwitterionic methionine residue plus a maleate anion. The backbone conformation angles Ψ¹ and Ψ² are in cis and trans configurations for both the methionine and methioninium residues, respectively. Amino and carboxyl groups of the methioninium and methionine residues are connected through N–H…O hydrogen bonds leading to a ring R₂²(10) motif.     

Review: Transition metal-based nanolamellar phases

This paper is the state-of-the-art review of the transition metal-based nanolamellar phases of the (h_mc_n)_k types (h - hexagonal close-packed motif; c - cubic close-packed motif; m, n, and k - integers). These nanolamellar phases are emerging materials, which possess extraordinary properties, as a result of their crystal structure, where planar defects (stacking faults and/or twins) are an inherent part of their crystal structure. On the other hand, the stacking faults and/or twins are nanostructuring elements, which define nanostructure-properties relationships. The thickness of nanolamella, which is formed by two parallel adjacent stacking faults on a nanometer scale, is ∼1 nm. The density of stacking faults can reach 10¹⁵ cm⁻³ that approach the density of grain boundaries in nanostructured materials. Formation of the nanolamellar phases is a result of structural phase transformation in consequence of ordering of interstitial C/N-atoms and vacancies in metalloid sub-lattice of the non-stoichiometric NaCl-type carbides/nitrides, resulting in the structural phase change associated with regular ordering of stacking. No any review has been published from that time of the discovery of the stunning phases, which are a novel segment of nanostructured materials. The aim of this paper is to advance the research, development, and applications of the transition metal-based nanolamellar phases.Undoubtedly the formation of the ζ-phases occurs as a result of regular removal of every fourth non-metal layer followed by shearing of transition metal layers to yield the sequence

…↑AγBαCβA↑CβAγBαC↑BαCβAγB↑…,     

Role of Redox‐Inactive Transition‐Metals in the Behavior of Cation‐Disordered Rocksalt Cathodes

Owing to the capacity boost from oxygen redox activities, Li‐rich cation‐disordered rocksalts (LRCDRS) represent a new class of promising high‐energy Li‐ion battery cathode materials. Redox‐inactive transition‐metal (TM) cations, typically d⁰ TM, are essential in the formation of rocksalt phases, however, their role in electrochemical performance and cathode stability is largely unknown. In the present study, the effect of two d⁰ TM (Nb⁵⁺ and Ti⁴⁺) is systematically compared on the redox chemistry of Mn‐based model LRCDRS cathodes, namely Li_1.3Nb_0.3Mn_0.4O₂ (LNMO), Li_1.25Nb_0.15Ti_0.2Mn_0.4O₂ (LNTMO), and Li_1.2Ti_0.4Mn_0.4O₂ (LTMO). Although electrochemically inactive, d⁰ TM serves as a modulator for oxygen redox, with Nb⁵⁺ significantly enhancing initial charge storage contribution from oxygen redox. Further studies using differential electrochemical mass spectroscopy and resonant inelastic X‐ray scattering reveal that Ti⁴⁺ is better in stabilizing the oxidized oxygen anions (O^n?, 0 < n < 2), leading to a more reversible O redox process with less oxygen gas release. As a result, much improved chemical, structural and cycling stabilities are achieved on LTMO. Detailed evaluation on the effect of d⁰ TM on degradation mechanism further suggests that proper design of redox‐inactive TM cations provides an important avenue to balanced capacity and stability in this newer class of cathode materials.     

Improved parametric time warping for proteomics

We present an improved version of parametric time warping, which enables the method to be used in LC-MS measurements in proteomics. The new features include a new similarity measure for comparing warped chromatograms, an insurance against peaks at the extremes of the chromatograms disappearing because of the warping, and the possibility to select and use multiple traces in searching the optimal alignment. Moreover, we present an alignment strategy combining global and individual alignments for aligning LC-MS data that exhibit different shifts within the same sample, at the same retention time. Using an LC-MS data set consisting of E. coli homogenates that were measured in several batches over a period of six months, we show the benefits of the improved algorithm and the merits of the new strategy. The algorithm is publicly available as the R package ptw.     

Coupling of the Nuclear Spin Alignment of Quadrupolar Nuclei to 3He at Interfaces

Measurements of the relaxation of nuclear spin alignments have been carried out using NQR spin echo studies of powdered antimony in contact with liquid ³ He. This system differs from previous studies of the relaxation and magnetization coupling of ³ He at surfaces because the magnetizations < S _z > 0. The spin alignments < S _z ² > are the relevant dynamical variables. The analysis of the experimental results shows that the coupling of the nuclear spin alignment of the antimony nuclei to the magnetization of the ³ He nuclei is determined by the spectral density of the fluctuations of the spin-spin dipolar couplings at the interface. This power density is attributed to the zero-point motions (atom-atom and atom-vacancy exchange) of the ³ He atoms on the surface of the metal particles.     

Optical and ESR investigation of borate glasses containing single and mixed transition metal oxides

The optical and electron spin resonance (ESR) spectra of barium borate glasses, containing the oxides of V, Fe and Cu separately and in mixed proportions, have been studied. The optical spectra of the single transition metal (TM) oxide glasses showed the usual features, while those for the mixed glasses showed single bands without showing individual features of the single TM oxide glasses. However, the linear plots of optical density against composition revealed the presence of two valence states for each TM element, and this was confirmed by ESR results as well. The ESR spectra of the mixed glasses showed a complicated interaction pattern for two different TM ions, in comparison with those of the glasses containing a single TM ion. For the Fe-V glasses, the progressively vanishing hyperfine structure of the VO²⁺ complex with increasing addition of iron oxide is discussed in terms of nuclear spin relaxation, cross-relaxation between two spin systems and spin diffusion within the vanadium spin system. The covalency of the VO²⁺ complex and the number of distorted Fe³⁺ ions were found to decrease with increasing addition of Fe₂O₃ replacing V₂O₅. Similar features were noted for the Cu-V glasses; the spectra of Cu-Fe glass also showed a strong interaction between two different TM ions. It has been suggested that all the possible four valence states (for a given mixed glass) from two different TM elements are present, and that pairing of two different TM ions from two dissimilar TM elements occurs, facilitating the formation of associates (e.g. V⁴⁺-O-Fe³⁺).     

Van der Waals force contribution to the vertical alignment of liquid crystal on Al2O3 films using ion-beam method

This study demonstrates liquid crystal (LC) alignment on Al₂O₃ films using the ion-beam (IB) method as well as the optimization of the IB irradiation condition. Uniform LC alignment was achieved at an IB incident energy of 1800 eV with an IB incident angle of 45°, while inferior LC alignments were observed in other tested conditions. The pretilt angles and transmittances of the LC cell were also shown as part of the same trend for the LC alignment states. This result was subject to van der Waals forces which were caused by topographical changes during the IB process.     

Thermomigration and electromigration in Sn8Zn3Bi solder joints

Individual effect of thermomigration (TM) and combined effect of TM and electromigration (EM) on the microstructural variation in Sn8Zn3Bi was investigated by stressing line-type Au/Ni–P/Cu-Sn8Zn3Bi-Au/Ni–P/Ni solder joints with a 5 × 10³ A/cm² alternating current (AC) or direct current (DC) at 110°C. Due to the different thermoelectric characteristics of Cu and Ni wires, a thermal gradient of 196°C/cm could be established across the solder joints according to the finite element simulation. In AC current stressing, there is no EM effect and only TM dominates the migration. Microstructural study shows that Zn atoms migrate towards the lower temperature side during TM. In DC current stressing, it is found that both EM and TM play important roles depending various experimental conditions. And the energy change during the EM and the TM is estimated to be ∆ω_em 3.2 × 10⁻²⁸ Joule and ∆ω_em 2.2 × 10⁻²⁸ Joule, respectively. Upon different current directions in DC current stressing, there is a counteractive or accelerated effect between TM and EM on Zn migration, resulting different microstructures at the cathode side in the solder joints.     

Multiple alignment through protein secondary-structure information

It is well known that protein secondary-structure information can help the process of performing multiple alignment, in particular when the amount of similarity among the involved sequences moves toward the "twilight zone" (less than 30% of pairwise similarity). In this paper, a multiple alignment algorithm is presented, explicitly designed for exploiting any available secondary-structure information. A layered architecture with two interacting levels has been defined for dealing with both primary- and secondary-structure information of target sequences. Secondary structure (either available or predicted by resorting to a technique based on multiple experts) is used to calculate an initial alignment at the secondary level, to be arranged by locally scoped operators devised to refine the alignment at the primary level. Aimed at evaluating the impact of secondary information on the quality of alignments, in particular alignments with a low degree of similarity, the technique has been implemented and assessed on relevant test cases.