Search

Excitatory amino acid transporters (EAATs) remove glutamate from synapses. They maintain an efficient synaptic transmission and prevent glutamate from reaching neurotoxic levels. Glutamate transporters couple the uptake of one glutamate to the cotransport of three sodium ions and one proton and the countertransport of one potassium ion. The molecular mechanism for this coupled uptake of glutamate and its co- and counter-transported ions is not known. In a crystal structure of the bacterial glutamate transporter homolog, [Glt.sub.ph], only two cations are bound to the transporter, and there is no indication of the location of the third sodium site. In experiments using voltage clamp fluorometry and simulations based on molecular dynamics combined with grand canonical Monte Carlo and free energy simulations performed on different isoforms of [Glt.sub.ph] as well on a homology model of EAAT3, we sought to locate the third sodium-binding site in EAAT3. Both experiments and computer simulations suggest that T370 and N451 (T314 and N401 in [Glt.sub.ph]) form part of the third sodium-binding site. Interestingly, the sodium bound at T370 forms part of the binding site for the amino acid substrate, perhaps explaining both the strict coupling of sodium transport to uptake of glutamate and the ion selectivity of the affinity for the transported amino acid in EAATs. excitatory amino acid transporters | fluorescence | the sodium/aspartate symporter from Pyrococcus horikoshii ([Glt.sub.ph]) | simulations doi/ 10.1073/pnas.1006289107

The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB. One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (~53-kDa and ~47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a [K.sub.m] value for PABA-GLU of 60 [+ or -] 0.08 [micro]M and a specific activity of 63,300 [+ or -] 600 nmol min-1 mg-l. Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway. doi: 10.1128/JB.01362-09

Glutamate transporters regulate synaptic concentrations of this neurotransmitter by coupling its flux to that of sodium and other cations. Available crystal structures of an archeal homologue of these transporters, GItPh, resemble an extracellular-facing state, in which the bound substrate is occluded only by a small helical hairpin segment called HP2. However, a pathway to the cytoplasmic side of the membrane is not clearly apparent. We previously modeled an alternate state of a transporter from the neurotransmitter:sodium symporter family, which has an entirely different fold, solely on the presence of inverted-topology structural repeats. In GItPh, we identified two distinct sets of inverted-topology repeats and used these repeats to model an inward-facing conformation of the protein. To test this model, we introduced pairs of cysteines into the neuronal glutamate transporter EAAC1, at positions that are >27 [Angstrom] apart in the crystal structures of GItPh, but [approximately equal to] 10 [Angstrom] apart in the inward-facing model. Transport by these mutants was activated by pretreatment with the reducing agent dithithreitol. Subsequent treatment with the oxidizing agent copper(II)(1,10-phenantroline)3 abolished this activation. The inhibition of transport was potentiated under conditions thought to promote the inward-facing conformation of the transporter. By contrast, the inhibition was reduced in the presence of the nontransportable substrate analogue D,L-threo-[beta]-benzyloxyaspartate, which favors the outward-facing conformation. Other conformation-sensitive accessibility measurements are also accommodated by our inward-facing model. These results suggest that the inclusion of inverted-topology repeats in transporters may provide a general solution to the requirement for two symmetry-related states in a single protein. alternating access | conformationally sensitive cross-linking | homology modeling | neurotransmitter | secondary transport doi/10.1073/pnas.0908570106

The activity of many ligand-gated ion channels and cell surface receptors is modulated by small molecules and ions, but an understanding of the underlying molecular mechanisms is scarce. For kainate, but not AMPA subtype glutamate receptors, the binding of [Na.sup.+] and [Cl.sup.-] ions to discrete, electrostatically coupled sites in the extracellular ligand binding domain (LBD) dimer assembly regulates the rate of entry into the desensitized state, which occurs when the dimer interface ruptures and the channel closes. Studies on glutamate receptors have defined the LBD dimer assembly as a key functional unit that controls activation and desensitization. Here we use analytical ultracentrifugation to probe the energetic effects of allosteric ions on kainate receptor dimer stability in solution, using a GluR6 mutant that desensitizes slowly. Our results show that sodium and chloride ions modulate kainate receptor dimer affinity as much as 50-fold, and that removal of either [Cl.sup.-] or [Na.sup.+] disrupts the dimer. The applicability of a similar allosteric mechanism for modulation of delta2 glutamate receptors by [Ca.sup.2+] was also tested. Our results indicate that ions can contribute substantial free energy to active state stabilization in both these receptors, and provide quantitative measurements of the energetic consequences of allosteric ion binding to a ligand-gated ion channel. allosteric modulation | analytical ultracentrifugation | desensitization | ion channels | thermodynamics

Glutamate (Glu) is a major intestinal oxidative fuel, key neurotransmitter, and may be a useful dietary supplement to augment health of the infant gut. We quantified the metabolic fate of various supplemental dietary Glu intakes in young pigs surgically implanted with vascular, intraduodenal (ID), or intragastric (IG) catheters and a portal blood flow probe. Piglets were acutely fed a range of dietary Glu intakes using a basal milk formula (100%) supplemented with varying amounts of monosodium Glu (up to 400%) via ID or IG routes. We quantified the gastrointestinal metabolic fate of dietary Glu using [U-[sup.13]C] Glu tracer. The Glu net absorption in the basal 100% group was low in both ID and IG groups, ranging from 13 to 17% of intake. Enteral Glu supplementation significantly increased the absolute absorption rate and arterial concentration of Glu. In both the ID and IG groups, enteral [[sup.13]C]Glu absorption was limited (

AMPA-type ([alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) glutamate receptors (AMPARs) mediate post-synaptic depolarization and fast excitatory transmission in the central nervous system. AMPARs are tetrameric ion channels that assemble in the endoplasmic reticulum (ER) in a poorly understood process. The subunit composition determines channel conductance properties and gating kinetics, and also regulates vesicular traffic to and from synaptic sites, and is thus critical for synaptic function and plasticity. The distribution of functionally different AMPARs varies within and between neuronal circuits, and even within individual neurons. In addition, synapses employ channels with specific subunit stoichiometries, depending on the type of input and the frequency of stimulation. Taken together, it appears that assembly is not simply a stochastic process. Recently, progress has been made in understanding the molecular mechanisms underlying subunit assembly and receptor biogenesis in the ER. These processes ultimately determine the size and shape of the postsynaptic response, and are the subject of this review.

Self-referencing [H.sup.+] t-selective microelectrodes were used to measure extracellular proton fluxes from cone-driven horizontal cells isolated from the retina of tile catfish (Iclalurus punctatus). The neurotransmitter glutamate induced an alkalinization of the area adjacent to tile external face of the cell membrane. The etfect of glutamate occurred regardless of whether the external solution was buffered with l mM HEPES, 3 mM phosphate, or 24 mM bicarbonate. The AMPA/kainate receptor agonist kainate and the NMDA receptor agonist N-methyl-D-aspartate both mimicked the effect of glutamate. The effect of kainate on proton flux was inhibited by the AMPA/kainate receptor blocker CNQX, and the effect of NMDA was abolished by the NMDA receptor antagonist DAP-5. Metabotropic glutamate receptor agonists produced no alteration in proton fluxes from horizontal cells. Depolarization of cells either by increasing extracellular potassium or directly by voltage clamp also produced an alkalinization adjacent to the cell membrane. The effects of depolarization on proton flux were blocked by 10 [micro]M nifedipine, an inhibitor of L-type calcium channels. The plasmalemma [Ca.sup.2+]/[H.sup.+] ATPase (PMCA) blocker 5(6)-carboxyeosin also significantly reduced proton flux modulation by glutamate. Our results are consistent with the hypothesis that glutamate-induced extracellular alkalinizations arise from activation of tile PMCA pump following increased intra-cellular calcium entry into cells. This process might help to relieve suppression of photoreceptor neurotransmitter release that results from exocytosed protons from photoreceptor synaptic terminals. Our findings argue strongly against the hypothesis that protons released by horizontal cells act as the inhibitory feedback neurotransmitter that creates the surround portion of the receptive fields of retinal neurons.

Presynaptic ionotropic glutamate receptors are emerging as key players in the regulation of synaptic transmission. Here we identify GluR7, a kainate receptor (KAR) subunit with no known function in the brain, as an essential subunit of presynaptic autoreceptors that facilitate hippocampal mossy fiber synaptic transmission. [GluR7.sup.-/-] mice display markedly reduced short- and long-term synaptic potentiation. Our data suggest that presynaptic KARs are GluR6/ GluR7 heteromers that coassemble and are localized within synapses. We show that recombinant GluR6/GluR7 KARs exhibit low sensitivity to glutamate, and we provide evidence that presynaptic KARs at mossy fiber synapses are likely activated by high concentrations of glutamate, Overall, from our data, we propose a model whereby presynaptic KARs are localized in the presynaptic active zone close to release sites, display low affinity for glutamate, are likely [Ca.sup.2+]-permeable, are activated by single release events, and operate within a short time window to facilitate the subsequent release of glutamate. kainate receptors | presynaptic glutamate receptors | short-term plasticity | synaptic plasticity

Abstract The dopamine hypothesis of schizophrenia is the principal explanatory model of antipsychotic drug action. Recent discoveries extend our understanding of the neurochemistry of schizophrenia, with increasing evidence of dysfunction [...]

The analysis of cell signaling requires the rapid and selective manipulation of protein function. We have synthesized photoswitches that covalently modify target proteins and reversibly present and withdraw a ligand from its binding site due to photoisomerization of an azobenzene linker. We describe here the properties of a glutamate photoswitch that controls an ion channel in cells. Affinity labeling and geometric constraints ensure that the photoswitch controls only the targeted channel, and enables spatial patterns of light to favor labeling in one location over another. Photoswitching to the activating state places a tethered glutamate at a high (millimolar) effective local concentration near the binding site. The fraction of active channels can be set in an analog manner by altering the photostationary state with different wavelengths. The bistable photoswitch can be turned on with millisecond-long pulses at one wavelength, remain on in the dark for minutes, and turned off with millisecond long pulses at the other wavelength, yielding sustained activation with minimal irradiation. The system provides rapid, reversible remote control of protein function that is selective without orthogonal chemistry. azobenzene | ion channel | photoisomerization | optical switch | remote control

The mutations in either of the two conserved glutamic acid residues, E107 and E99, has resulted in the loss of quinol oxidase activity, resulting in the loss of the two hemes at the active site, hemes d and [b.sub.595]. Fourier transform infrared (FTIR) spectroscopy results have shown that E107 is protonated at pH 7.6 and that it is perturbed by the reduction of the heme d/heme [b.sub.595] binuclear center at the active site.

Two mechanisms are proposed for the racemization reaction catalyzed by Aquifex pyrophillus glutamate racemase enzyme. The results have shown that the behavior of an enzyme depends on the ionization state of its active site residues and justifies the calculation of their ionization probabilities to understand the enzyme catalytic mechanism.

Proteins whose synthesis is enhanced by polyamines at the level of translation were identified in a polyaminerequiring mutant cultured in the presence of 0.1% glucose and 0.02% glutamate instead of 0.4% glucose as an energy source. Under these conditions, enhancement of cell growth by polyamines was almost the same as that in the presence of 0.4% glucose. It was found that synthesis of RpoN, Cra, and H-NS was enhanced by polyamines at the level of translation at the early logarithmic phase of growth ([A.sub.540] of 0.15). The effects of polyamines on synthesis of RpoN, H-NS, and Cra were due to the existence of unusual Shine-Dalgarno sequences (RpoN and H-NS) and an inefficient GUG initiation codon (Cra) in their mRNAs. Thus, rpoN, cra and hns genes were identified as new members of the polyamine modulon. Because most of the polyamine modulon genes thus far identified encode transcription factors (RpoS [[[sigma].sup.38]], Cya, FecI [[[sigma].sup.18]], Fis, RpoN [[[sigma].sup.54]], Cra, and H-NS), DNA microarray analysis of mRNA expressed in cells was performed. At the early logarithmic phase of growth, a total of 97 species of mRNAs that were up-regulated by polyamines more than twofold were under the control of seven polyamine modulon genes mentioned above.

A combination of the growth probability method and several three-dimensional (3D) nucleation simulations is used to study the formation of polymorphs, wherein the nucleus size acts as a function of the driving force for crystallization. The study has found application in a large number of fields that includes a yellow isoxazolone dye, paracetamol and L-glutamic acid, as they show dominant nucleation of their stable form at all driving forces.

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.

A study was conducted to identify a second calmodulin-regulated site targeted by calpain in [alpha]II spectrin and three novel sites of cleavage in [beta]II spectrin. It was found that glutamate stimulation of cortical neurons preferentially induces [alpha]II spectrin cleavage as a very early event while in non-glutamate responsive SH-SY5Y cells, spectrin is unaffected by glutamate exposure.

A range of 2-acrylamidopyridines, showing subtle differences in solution binding toward carboxylic acids, has been investigated as functional monomers in molecular imprinting. Imprinting of N-Z-L-glutamic acid with one such monomer is shown to be effective in the creation of a molecularly imprinted polymer (MIP) with recognition properties for its template and also for larger molecules containing glutamic acid residues. In comparison to a MIP prepared via a more 'traditional' approach, the new polymeric receptors exhibit reduced nonspecific binding. The new receptors are compared with previously reported urea-based receptors targeting the glutamic acid residue and receptors targeting the pteridine substructure of folic acid.

Two compact denatured states of glutaminyl-tRNA synthetase (GInRS) under equilibrium conditions in the presence of a naturally occurring osmolyte, L-glutamate are characterized. Dynamic light scattering studies reveal that both the compact denatured states are aggregated under the measurement conditions.

The retinal L-type [Ca.sup.2+] channel Cav1.4 is distinguished from all other members of the high voltage-activated (HVA) [Ca.sup.2+] channel family by lacking [Ca.sup.2+]-calmodulin-dependent inactivation. In synaptic terminals of photoreceptors and bipolar cells, this feature is essential to translate graded membrane depolarizations into sustained [Ca.sup.2+] influx and tonic glutamate release. The sequences conferring [Ca.sup.2+]-dependent inactivation (CDI) are conserved throughout the HVA calcium channel family, raising the question of how Cav1.4 manages to switch off CDI. Here, we identify an autoinhibitory domain in the distal C terminus of Cav1.4 that serves to abolish CDI. We show that this domain (ICDI, inhibitor of CDI) uncouples the molecular machinery conferring CDI from the inactivation gate by binding to the EF hand motif in the proximal C terminus. Deletion of ICDI completely restores [Ca.sup.2+]-calmodulin-mediated CDI in Cav1.4. CDI can be switched off again in the truncated Cav1.4 channel by coexpression of ICDI, indicating that ICDI works as an autonomous unit. Furthermore, we show that in the Cav1.2 L-type [Ca.sup.2+]-channel replacement of the distal C terminus by the corresponding sequence of Cav1.4 is sufficient to block CDI. This finding suggests that autoinhibition of CDI can be introduced principally into other [Ca.sup.2+] channel types. Our data provide a previously undescribed perspective on the regulation of HVA calcium channels by [Ca.sup.2+]. calmodulin | Cav1.4 | retina | Ca channels

The accumulation of compatible solutes is studied in the hyperthermophilic bacterium Aquifex pyrophilus as a function of the temperature and the NaCl concentration of the growth medium. The results reveal the presence of alpha- and beta-glutamate, di-mannosyl-di-myo-inositol phosphate, di-myo-imositol phosphate, and a compound identified as 1-glyceryl-1-myo-insoityl phosphate in living systems.

Five Glu 192 mutants of PCP-0SH (where PSCP= pyrrolidone carboxyl peptidases) from Pyrococcus furiosus were constructed and their thermal and pH-induced unfolding and crystals structures were compared in order to explain the role of the buried glutamic acid in stabilizing pyrrolidone carboxyl peptidases (PCP) from hyperthermophiles. The results showed that completely buried Glu 192 contributes to the stabilization of PCP-0SH because of the formation of strong intramolecular hydrogen bonds and these hydrogen bonds contribute more to the conformational stability of proteins.

A description is provided of stereocontrolled and convergent total synthesis of amphidinolide T3. The construction of a retrosynthetic scheme led to the recognition of readily available and enantiomerically related compounds as starting materials for the total synthesis of amphidinolide T3.

In cerebral circulation, epileptic seizures associated with excessive release of the excitatory neurotransmitter glutamate cause endothelial injury. Heme oxygenase (HO), which metabolizes heine to a vasodilator, carbon monoxide (CO), and antioxidants, biliverdin/bilirubin, is highly expressed in cerebral microvessels as a constitutive isoform, HO-2, whereas the inducible form, HO-1, is not detectable. Using cerebral vascular endothelial cells from newborn pigs and HO-2-knock-out mice, we addressed the hypotheses that 1) glutamate induces oxidative stress-related endothelial death by apoptosis, and 2) HO-1 and HO-2 are protective against glutamate cytotoxicity. In cerebral endothelial cells, glutamate (0.1-2.0 mM) increased formation of reactive oxygen species, including superoxide radicals, and induced major keystone events of apoptosis, such as NF-[kappa]B nuclear translocation, caspase-3 activation, DNA fragmentation, and cell detachment. Glutamate-induced apoptosis was greatly exacerbated in HO-2 gene-deleted murine cerebrovascular endothelial cells and in porcine cells with pharmacologically inhibited HO-2 activity. Glutamate toxicity was prevented by superoxide dismutase, suggesting apoptotic changes are oxidative stress related. When HO-1 was pharmacologically upregulated by cobalt protoporphyrin, apoptotic effects of glutamate in cerebral endothelial cells were completely prevented. Glutamate- induced reactive oxygen species production and apoptosis were blocked by a CO-releasing compound, CORM-Al (50 [micro]M), and by bilirubin (1 [micro]M), consistent with the antioxidant and cytoprotective roles of the end products of HO activity. We conclude that both HO-1 and HO-2 have anti-apoptotic effects against oxidative stressrelated glutamate toxicity in cerebral vascular endothelium. Although HO-1, when induced, provides powerful protection, HO-2 is an essential endogenous anti-apoptotic factor against glutamate toxicity in the cerebral vascular endothelium. endothelium; carbon monoxide; bilirubin; injury; reactive oxygen species; heine oxygenase

A short enantioselective synthesis of (2R)-hydroxymethyl glutamic acid (HMG) is developed starting from Garner's aldehyde using an alkylidene carbene 1,5-CH insertion as a method to construct the quaternary stereocenter. A variety of conditions are examined for the oxidative cleavage of the key cyclopentene intermediate and it is found that RuCI3/NaIO4 led to the desired amino bis-acid product.

Enzymatic hydroxylation reactions carried out by 2-oxoglutarate (2OG) dependent iron-containing oxygenases were implicated in oxygen sensing. Cobalt and nickle are capable of inducing hypoxic stress in cells in addition to oxygen depletion, by inhibiting oxygenase activity.

The significance of three structural regions, namely the N-terminal pyroglutamate residue, the hydrophobic cluster between helix I and the first beta-sheet, and the C-terminal disulfide bond, which exhibit structural peculiarities in comparison to ribonuclease A (RNase A), is probed for the stability of onconase (ONC) to temperature and guanidine hydrochloride induced denaturation. A dramatic deceleration of the unfolding reaction is the cause of the strong stabilization of ONC in comparison to RNase A.

The cloning, expression and characterization of the protein Pa5106 from Pseudomonas aeruginosa, along with two other proteins from this same organism, Pa5091 and Pa3175, are reported. The results show that Pa5016 catalyzes the deimination of N-formimino-L-glutamate to ammonia and N-formyl-L-glutamate, while Pa5091 catalyzes the hydrolysis of N-formyl-L-glutamate to formate and L-glutamate, and protein Pa3175 catalyze the hydrolysis of N-formimino-L-glutamate to formamide and L-glutamate.

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

Computer simulations on a QM/MM potential energy surface are carried out to gain insights into the catalytic mechanism of glutamate racemase (Murl). Studies suggest two possible roles for Murl as a catalyst, which are achieved without resorting to covalent bond formulation between the enzyme or cofactor and the transition state.

Ab-initio electronic structure methods are used to explore potential energy profiles pertinent to the fragmentation of gas-phase radicals to be formed in the new negative-ion mode (EDD ) electron-detachment dissociation mass spectroscopic studies of peptides. It is found that fragmentation of nitrogen-centered radicals formed in EDD results in cleavage to produce so-called a(dot)/x fragments rather than a/x(dot) fragments because producer former involves smaller barrier and is thermoneutral.

The effect of salinity and growth temperature on the accumulation of intracellular organic solutes is determined in the hyperthermophilic archeon Palaeococcus ferrophilus. The osmotic and heat stress responses of Palaeococcus ferrophilus in regard to compatible solute accumulation are described, the genes for the synthesis of mannosylglycerate (MG) are identified and the recombinant proteins are characterized.

An efficient synthesis of three diastereomers of 9-tert-butoxycarbonyl-2-oxo-3-(N-tert-butoxycarbonylamino)-1-azabicyclo[4.3.0]nonane is described. Acidolytic deprotection after Fmoc-protection provided building blocks suitable for solid-phase synthesis.

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.

A D165N mutant of clostridial glutamate dehydrogenase (GDH) in which Asn replaces the catalytic Asp, has showed a residual 2% of wild-type activity when purified after expression in Escherichia coli at 37 degree centigrade. The D165N mutant is at least 70,000 times less active than the wild-type enzyme, but deamidation restores the natural catalytic aspartate and with it the activity of the protein, in a chemical reversion at the protein level of a mutation at the DNA level.

An attempt is made to show that structure-based mutation L651V converts the GluR4 AMPA receptor into a dual-specificity AMPA/kinate receptor fully activated by both agonists. Further, the correlation between agonist efficacy and a series of vibrational and fluorescence spectroscopic signals of agonist binding to the corresponding wild-type and mutant GluR4 ligand-binding domains is investigated.

The X-ray crystal structure of gama-carboxyglutamic acid (Gla) domains is established. It is found that these domains are stable to long all-atoms simulations in explicit solvent.

The Erwinia chrysanthemi 3937 endogenous osmolytes were analyzed and showed that the bacterium does not synthesize glutamine and alpha-glucosylglycerate in enterobacteria. The study showed that it is not true only halophilic organisms and its counterpart glucosylglycerol accumulate that glucosylglycerate by nonhalophilic ones.

Studies done on the role of glutamic acid 181 in the bovine rhodopsin retinylidene chromophore pocket by expressing E181 mutants in COS cells and measuring as a function of time, the absorbance changes produced after excitation of lauryl maltoside pigment suspensions with 7 ns laser pulses is presented. The result is consistent with the idea that a negative charge at position 181 stabilizes protonation of the retinylidene Schiff base in the later intermediates.

The potential contribution of posttranslational modifications, in particular glutamylation of the C-termini of the tubulin chains, to the cold-assembling phenotype of the microtubules of Antarctic fishes is investigated. It is suggested that the unique residue substitutions in the primary structures of Antarctic fish tubulin isotypes and quantitative changes in isoform glutamylation act synergistically to adapt microtubule assembly to low temperatures.

The comparative genomic methods to map available mammalian thioredoxin reductase 1 (TR1) cDNA sequences and identified novel, species-specific and common alternatively spliced forms of this protein is described. Analysis of these forms revealed the basic TR1 module is fused to various redox sequences and that such fusion allows the linkage of these sequences to an electron donor, NADPH, and provides the expression of the fusion proteins in temporal and spatial manner.

The pK(sub a) values of all of the titratable residues in rhodospin are first calculated and then the effect of moving the lipid/water interface on both sides of rhodospin is evaluated to test the hypothesis that the protonation of glutamic acid E134 is particularly affected by its position relative to the hydrophobic region of the membrane. The results show that the protonation state of E 134 is sensitive to its environment.

The formation of interpolyelectrolyte complex (PEC) between sodium poly(l-glutamate) and fourth generation aminoterminated poly(amido amine) dendrimer and its aggregation by static and dynamic light scattering is discussed. It is observed for systems with carboxylate excess over amino/ammonium groups and again attributable to compact particles with similar size to the former.

The study presents a report on the triple nuclear magnetic resonance pulse scheme, which is useful in measuring aspartic and glutamic acid side-chains. The study correlates side-chain carboxyl carbon chemical shifts in these residues.