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Metabotropic glutamate receptors play major roles in the activation of excitatory synapses in the central nerve system. We determined the crystal structure of the entire extracellular region of the group II receptor and that of the ligand-binding region of the group III receptor. A comparison among groups I, II, and III provides the structural basis that could account for the discrimination of group-specific agonists. Furthermore, the structure of group II includes the cysteine-rich domain, which is tightly linked to the ligand-binding domain by a disulfide bridge, suggesting a potential role in transmitting a ligand-induced conformational change into the downstream transmembrane region. The structure also reveals the lateral interaction between the two cysteine-rich domains, which could stimulate clustering of the dimeric receptors on the cell surface. We propose a general activation mechanism of the dimeric receptor coupled with both ligand-binding and interprotomer rearrangements. crystallization | cysteine-rich region | ligand binding | G protein-coupled receptor

Glutamyl-tRNA (Glu-tRNA), formed by Glu-tRNA synthetase (GluRS), is a substrate for protein biosynthesis and tetrapyrrole formation by the Cs pathway, in this route Glu-tRNA is transformed to [delta]-aminolevulinic acid, the universal precursor of tetrapyrroles (e.g., heme and chlorophyll) by the action of Glu-tRNA reductase (GluTR) and glutamate semialdehyde aminotransferase. GluTR is a target of feedback regulation by heme. In Acidithiobacillus ferrooxidans, an acidophilic bacterium that expresses two GluRSs (GluRS1 and GluRS2) with different tRNA specificity, the intracellular heme level varies depending on growth conditions. Under high heme requirement for respiration increased levels of GluRS and GluTR are observed. Strikingly, when intracellular heme is in excess, the cells respond by a dramatic decrease of GluRS activity and the level of GluTR. The recombinant GluRS1 enzyme is inhibited in vitro by hemin, but NADPH restores its activity. These results suggest that GluRS plays a major role in regulating the cellular level of heme. gene expression | protein biosynthesis | tetrapyrrole biosynthesis

A study demonstrates the regulation of the channel opening process by the alternate splicing method, which generates flip and flop variants of GluR3 receptors by controlling the rate of the channel closing. The analysis presents the differences in the channel opening kinetics of the [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), which may also be extensively used for future studies.

The status of the N-terminus of proteins is important for amino acid sequencing by Edman degradation, protein identification by shotgun and top-down techniques, and to uncover biological functions, which may be associated with modifications. In this study, we investigated the pyroglutamic acid formation from N-terminal glutamic acid residues in recombinant monoclonal antibodies. Almost half the antibodies reported in the literature contain a glutamic acid residue at the N-terminus of the light or the heavy chain. Our reversed-phase high-performance liquid chromatography-mass spectrometry method could separate the pyroglutamic acid-containing light chains from the native light chains of reduced and alkylated recombinant monoclonal antibodies. Tryptic peptide mapping and tandem mass spectrometry of the reduced and alkylated proteins was used for the identification of the pyroglutamic acid. We identified the formation of pyroglutamic acid from N-terminal glutamic acid in the heavy chains and light chains of several antibodies, indicating that this nonenzymatic reaction does occur very commonly and can be detected after a few weeks of incubation at 37 and 45 [degrees]C. The rate of this reaction was measured in several aqueous buffers with different pH values, showing minimal formation of pyroglutamic acid at pH 6.2 and increased formation of pyroglutamic acid at pH 4 and pH 8. The half-life of the N-terminal glutamic acid was ~9 months in a pH 4.1 buffer at 45 [degrees]C. To our knowledge, we showed for the first time that glutamic acid residues located at the N-terminus of proteins undergo pyrogiutamic acid formation in vitro.

Silent synapses, or excitatory synapses that lack functional [alpha]-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), are thought to be critical for regulation of neuronal circuits and synaptic plasticity. Here, we report that SynGAP, an excitatory synapse-specific RasGAP, regulates AMPAR trafficking, silent synapse number, and excitatory synaptic transmission in hippocampal and cortical cultured neurons. Overexpression of SynGAP in neurons results in a remarkable depression of AMPAR-mediated miniature excitatory postsynaptic currents, a significant reduction in synaptic AMPAR surface expression, and a decrease in the insertion of AMPARs into the plasma membrane. This change is specific for AMPARs because no change is observed in synaptic NMDA receptor expression or total synapse density. In contrast to these results, synaptic transmission is increased in neurons from SynGAP knockout mice as well as in neuronal cultures treated with SynGAP small interfering RNA. In addition, activation of the extracellular signal-regulated kinase, ERK, is significantly decreased in SynGAP-overexpressing neurons, whereas P38 mitogen-activated protein kinase (MAPK) signaling is potentiated. Furthermore, ERK activation is up-regulated in neurons from SynGAP knockout mice, whereas P38 MAPK function is depressed. Taken together, these data suggest that SynGAP plays a critical role in the regulation of neuronal MAPK signaling, AMPAR membrane trafficking, and excitatory synaptic transmission. trafficking | Ras | glutamate | receptor I plasticity

The metabotropic glutamate receptor 1[alpha] (mGluR1[alpha]) is crucial for some forms of synaptic plasticity, by inducing various cell responses via coupling to various types of G proteins. Upon glutamate binding, an actie conformation, closed-open/active, of the extracellular domain is stabilized, which induces dimeric rearrangement in the intracellular domains, resulting in the initiation of downstream signals. We have confirmed that mGluR1[alpha] functionally interacts with both Gq and Gs pathways; a combination of fluorescent indicators showed that glutamate increased intracellular [Ca.sup.2+] and cAMP concentration ([[[Ca.sup.2+]].sub.i] and [[cAMP].sub.i]). By contrast, [Gd.sup.3+], a different type of ligand whose recognition site on mGluR1[alpha] is distinct from the glutamate site, increased only [[[Ca.sup.2+]].sub.i] and the concentration-activation curve was bell-shaped. FRET analysis revealed that a low concentration of [Gd.sup.3+] induced dimeric rearrangement of the intracellular domains of mGluR1[alpha] as does glutamate, whereas a high concentration of [Gd.sup.3+] reversed the FRET efficiency, which was consistent with a bell-shaped relationship between concentration and Gq activation. These results suggest that [Gd.sup.3+] induces an active and a sort of 'inactivated' conformation in mGluR1[alpha]. The [Gd.sup.3+]-induced active state is considered to correspond to the closed-closed/active conformation, revealed by previous x-ray crystallographic studies. In conclusion, the glutamate-induced closed-open/active state coupled both to Gs and Gq proteins whereas the [Gd.sup.3+]-induced closed-closed/active conformation state preferred Gq to Gs, suggesting that mGluR1[alpha] serves not only as a simple on/off switch but also as a multiple signaling path regulator. FRET | G protein-coupling receptor

A general and highly efficient asymmetric synthesis of potent and selective bicyclo[3.1.0]hexanes mGluR2/3 agonists is described. Densely functionalized, enantiomerically pure, monoprotected diols were prepared through a Et3Al-mediated intramolecular cyclopropanation with respect H or F endo selectivity in excellent yields.

Asymmetric Michael addition of chiral 2-fluoroenaminoesters derived from (S)-1-phenylethylamine to [alpha]-substituted methyl acrylate which leads to diastereomeric [gamma]-substituted [gamma]-fluoroglutamate precursors is reported. The results from [auper 19]F and [super 1]H NMR experiments and ab initio computations demonstrated the Michael addition diastereoselectivity for the asymmetric conjugate addition of fluorinated chiral [beta]-enaminoester to methyl [alpha]-acetamidoacrylate.

Steady-state kinetic, isotope transfer and positional isotope exchange experiments are used for studying a series of site-directed asparagine synthetase B (AS-B) mutants from Escherichia coli in which glutamate residue (Glu-348) is substituted by conservative amino acid replacements. The similarity of the chemistry catalyzed by AS-B, [beta]-lactam synthetase (BLS) and carbapenam synthetase (CPS) has highlighted the difficulty of predicting the functional outcome of single site mutations on enzymes that has catalyzed almost identical chemical transformations.

CBz-protected glutamic anhydride and Boc-protected o-aminobenzyl amine were utilized as starting material to carry out first total synthesis of structure of auranthine. The key steps in the synthesis include an intramolecular aza-Wittig reaction involving a lactam carbonyl group that delivered the diazepine core unit.

The synthesis and self-assembly of the new class of peptide chimera with a series of amphiphilic polypeptide-b-designed peptides are described in which the hydrophobic block is poly([gamma]-benzyl L-glutamate) (PBLG) and the hydrophilic block is a coiled-coil forming peptide (E). The studies have shown that as the structures have encapsulated water-soluble compounds and the surfaces are functionalized by the coiled-coil binding, these peptide-based nanocapsules have acted as delivery vehicles to specific targets in the body.

A study related to the synergistic regulation of the tryptophan synthase (TRPS) complex as a model of allosteric regulation and substrate channeling within protein complexes is presented. The atomically detailed dynamics and conformational changes of TRPS in the absence and presence of substrates is also demonstrated by using molecular dynamics simulations.

The in vitro properties of the C-truncated Escherichia coli GluRSs and their N-truncated complements GluRSs were characterized to understand the binding mechanism between ATP and the proper site of glutamate activation in Thermus thermophilus GluRS which requires the presence of [tRNA.sup.Glu]. The results confirmed the modular nature of GluRS and supported the model of a erecentE fusion of domains 4 and 5 to a proto-GluRS containing the catalytic domain and were able to recognize its tRNA substrate(s).

Two different linkers for pyrene attachment, namely 1-pyrenemethylamine (PMA) and 1-pyrenebutylamine (PBA) were used to synthesize two pyrene-labeled poly(glutamic acid) (Py-PGA). The method used provides insight into the diffusional encounters between pyrene pendants along the helix which could be used to study side-chain dynamics of structured polypeptides.

The properties of two site-specific mutants of human manganese superoxide dismutase (MnSOD) in which Glu162 is replaced with Asp (E162D) and Ala (E162A) are examined. The results have shown that Glu162 at the tetrameric interface in human MnSOD has supported stability and efficient catalysis and has an important role in regulating product inhibition.

An efficient synthesis of dysiherbaine with ionotropic glutamate receptors and its analogues is described. The structure and reactivity studies showed that the stereochemistry of C9 hydroxy group is responsible for the binding with glutamate receptors.

Atomic force microscopy (AFM)-based single molecule force microscopy is used for studying the interaction between poly(L-glutamic acid) (PLE) and calcite crystals. The PLE-calcite interaction is examined as a function of pH as well as the calcium concentration of the aqueous solution.

A computational study to discuss the reaction mechanism of the K-dependent vitamin, glutamate carboxylase is conducted.

A series of 12 monomeric alpha-helical peptides containing potential Glu-Xaa (i, i+3), (i, i+4) and (i, i+5) interactions are studied. Incorporating side chains just one methylene shorter resulted in significant pH dependence or lack of interaction.

The temperature dependence of steady state and pre-steady state kinetics of substrate transport is determined by the neuronal glutamate transporter excitatory amino acid carrier 1 (EAAC1) in order to understand the diffusional processes/conformational changes. The results have indicated that two conformational changes accompany glutamate translocation and at least one conformational change accompanies the relocation of the empty transporter.

The examination of poly-[gamma]-glutamate (PGA) productivity of Bacillus megaterium has shown NaCl-responsive PGA production in the bacterium. It is shown that salt plays a vital role in Bacillus megaterium in controlling the yield, molecular size and stereochemistry of bionylon.

A combination of neutron diffraction augmented with isotopic substitution and computer modeling using empirical potential structure refinement is used to extract detailed structural information for L-glutamic acid dissolved in 2 M NaOH solution. This method of extracting information from solution on an atomic scale is well proven for other liquids but has not previously been used to determine correlations in a system containing amino acids in solution.

A study was conducted to investigate the reaction of glutamate mutase with the glutamate analogue, 2-thiolglutarate. The present experiments demonstrate that 2-thioglutarate does react with glutamate to generate cobalamin II (CbI(II)) and an organic radical, which is probably the thioglycolate radical that arises by fragmentation of the initially formed 2-thiolglutaryl radical.

The biosynthesis of the calcium-dependent antibiotics (CDAs) is investigated. Cads and daptomycin, along with the related A54145 lipopeptides, all poses N-terminal fatty acid side chains and share several common amino acids, including acidic residues Asp, Glu, and 3-methylglutamic acid, some of which are conserved at the same relative positions in the decapeptide lactone cores.

A concise synthesis of neodysiherbaine A, and its structural analogues is described. An investigation of the described preliminary structure-activity relationship studies reveals that the alpha-oriented C9 hydro group is a critical element required for selective binding to the GluR5 kainate receptor subunit.

A demonstration of a new and simple method for the sonochemical formation of biocompatible core-shell microspheres is presented. These polyglutamate microspheres are highly stable throughout the pH, ionic strength, and temperature ranges encountered in vivo and the stability of polyglutamate microspheres appears to be due to hydrogen bonding networks and not covalent cross-linking.

A variety of high-level electronic structure calculations on two moderately sized organic molecules are performed and considerable sensitivity of the intramolecular potential energy surface to the method employed are found. It is shown by a novel analysis that the intramolecular equivalent of basis set superposition error competes with the errors in the intramolecular dispersion in causing this sensitivity.

A molecular modeling approach is used to study the stability of different polymorphic forms of L-glutamic acid through building and optimising molecular clusters of different sizes and shapes with the latter corresponding to the predicted crystal growth morphologies. The results reveal that the initially nucleating metastable alpha form is the more energetically stable form at small cluster sizes of ca. 200 molecular units, whereas the stable beta form is more stable when the cluster size is larger.

Glutamate transporters are thought to be assembled as trimers of identical subunits that line a central hole, possibly the permeation pathway for anions. The effect of multimerization on the transporter function is tested by coexpressing excitatory amino acid carrier 1(EAAC1)(sub WT) with the mutant transporter EAAC1(sub R446Q).

The theoretical analysis of the ligand-protein interactions in the binding pocket of the S1S2 domain of the ionotropic glutamate receptors in the closed conformation is reported. A detailed review of the interactions in the binding site is inferred by analyzing contributions to vibrational frequencies produced by protein residues forming the ligand-binding pocket.

The study produced wild-type recombinant myeloperoxidase (recMPO) and the variant Glu242Gln in Chinese hamster ovary cells and elucidated the role of the Glu2420heme ester linkage in the individual reaction steps of both the halogenation and peroxidase cycle. Disruption of the Glu2420heme ester linkage increases the flexibility of the prothetic group, thereby decreasing the rate of reaction of H2O2 and cyanide wit ferric MPO as well oxidation reactions of both (pseudo)halides and aromatic electron donors.