Amino acid availability in ruminants of cereals and cereal co-products

Microbial corrected in situ estimatesof the ruminal undegraded fraction (RU) and intestinal effectivedigestibility (IED) of amino acids (AA), except tryptophan, of rye, wheat and corn grains, wheat bran, wheat and barley distilled dried grains and corn gluten feed were measured on three rumen- and duodenum-cannulated wethers using 15N-labelling techniques and considering ruminal rates of particle comminution and outflow.

Protein diagenesis in Patella shells: implications for amino acid racemisation dating

Se analiza la racemización de aminoácidos en proteínas inter e intracristalinas en conchas de Patella y su utilización como herramienta geocronológica, fundamentalmente empleadas en yacimientos arqueológicos.The inter- and intra-crystalline fractions of Patella vulgata limpets recovered from archaeological sites in Northern Spain (covering Neolithic, Mesolithic, Magdalenian, Solutrean, and Aurignacian periods) were examined for amino acid composition and racemisation over time. The calcitic apex and rim areas of the shells were found to probably be composed of similar proteins, as the D/L values and amino acids were comparable and varied in the same way with increasing age; however, the mineral structures present in these areas differed. The aragonitic intermediate part of the shell showed a distinctly different amino acid composition and mineral structure. The main protein leaching from the inter-crystalline fraction occurred within the first 6000 yr after the death of the organism. In contrast, the intra-crystalline fraction — comprised of a different protein composition than the inter-crystalline fraction — appeared to behave as a closed system for at least 34 ka, as reflected by the lack of a significant decrease in the amino acid content; however, changes in the amino acid percentages occurred during this period. The concentration of aspartic acid remained almost constant with age both in inter- and intra-crystalline proteins, and its contribution to the total amino acid content increased with age at the expense of other amino acids such as glutamic acid, serine, glycine and alanine. Temperature is thought to play a key role in the amino acid racemisation of P. vulgata and could explain why in the localities belonging to the Gravettian and Solutrean period, which formed during relatively cold conditions, D/L values were similar to those detected in shells from sites formed during the Magdalenian.

Amino acid epimerization dating of coastal neotectonic processes in southeastern Iberian Peninsula: the region between Aguas and Antas river mouths

The coastal area between the mouths of the Aguas and Antas Rivers presents a deformed system of raised marine deposits, some of which have been strongly affected by active tectonics. The use of amino acid epimerization dating of Glycymeris shells from raised coastal deposits allowed determining the age of these marine deposits, all of them linked to highstand sea levels in the Mediterranean realm, with ages between MIS 11 and MIS 1. These results allowed corroborating the age of some previously studied sites, and using new sampling sites, the general aminostratigraphy for the Quaternary raised marine deposits on the Mediterranean coast was confirmed. The main deformation event took place after MIS 11 and continued until MIS 5, and was linked to the activity of the Palomares Faul

A synthetic all d-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 recognizes the wild-type fusion peptide in the membrane and inhibits HIV-1 envelope glycoprotein-mediated cell fusion

Recent studies demonstrated that a synthetic fusion peptide of HIV-1 self-associates in phospholipid membranes and inhibits HIV-1 envelope glycoprotein-mediated cell fusion, presumably by interacting with the N-terminal domain of gp41 and forming inactive heteroaggregates [Kliger, Y., Aharoni, A., Rapaport, D., Jones, P., Blumenthal, R. & Shai, Y. (1997) J. Biol. Chem. 272, 13496–13505]. Here, we show that a synthetic all d-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 (D-WT) of HIV-1 associates with its enantiomeric wild-type fusion (WT) peptide in the membrane and inhibits cell fusion mediated by the HIV-1 envelope glycoprotein. D-WT does not inhibit cell fusion mediated by the HIV-2 envelope glycoprotein. WT and D-WT are equally potent in inducing membrane fusion. D-WT peptide but not WT peptide is resistant to proteolytic digestion. Structural analysis showed that the CD spectra of D-WT in trifluoroethanol/water is a mirror image of that of WT, and attenuated total reflectance–fourier transform infrared spectroscopy revealed similar structures and orientation for the two enantiomers in the membrane. The results reveal that the chirality of the synthetic peptide corresponding to the HIV-1 gp41 N-terminal sequence does not play a role in liposome fusion and that the peptides’ chirality is not necessarily required for peptide–peptide interaction within the membrane environment. Furthermore, studies along these lines may provide criteria to design protease-resistant therapeutic agents against HIV and other viruses.

Synthetic amino acid copolymers that bind to HLA-DR proteins and inhibit type II collagen-reactive T cell clones

Copolymer 1 [poly(Y,E,A,K)] is a random synthetic amino acid copolymer of l-tyrosine, l-glutamic acid, l-alanine, and l-lysine that is effective both in suppression of experimental allergic encephalomyelitis and in the treatment of relapsing forms of multiple sclerosis. Copolymer 1 binds promiscuously and very efficiently to purified HLA-DR molecules within the peptide-binding groove. In the present study, YEAK and YEAK-related copolymers and type II collagen (CII) peptide 261–273, a candidate autoantigen in rheumatoid arthritis (RA), competed for binding to RA-associated HLA-DR molecules encoded by DRB1*0101 and DRB1*0401. Moreover, these copolymers (particularly YEAK, YAK, and YEK) inhibited the response of DR1- and DR4-restricted T cell clones to the CII epitope 261–273 by >50%. This direct evidence both for competitive interactions of these copolymers and CII peptide with RA-associated HLA-DR molecules and for inhibition of CII-specific T cell responses suggests that these compounds should be evaluated in animal models for rheumatoid arthritis.

Isolation and characterization of an amino acid-selective channel protein present in the chloroplastic outer envelope membrane

The reconstituted pea chloroplastic outer envelope protein of 16 kDa (OEP16) forms a slightly cation-selective, high-conductance channel with a conductance of Λ = 1,2 nS (in 1 M KCl). The open probability of OEP16 channel is highest at 0 mV (Popen = 0.8), decreasing exponentially with higher potentials. Transport studies using reconstituted recombinant OEP16 protein show that the OEP16 channel is selective for amino acids but excludes triosephosphates or uncharged sugars. Crosslinking indicates that OEP16 forms a homodimer in the membrane. According to its primary sequence and predicted secondary structure, OEP16 shows neither sequence nor structural homologies to classical porins. The results indicate that the intermembrane space between the two envelope membranes might not be as freely accessible as previously thought.

The interaction of the general anesthetic etomidate with the γ-aminobutyric acid type A receptor is influenced by a single amino acid

The γ-aminobutyric acid type A (GABAA) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (α1–6, β1–3, γ1–3, δ1, and ɛ1). Positive allosteric modulation of GABAA receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABAA receptors is uniquely dependent upon the β subunit subtype present within the receptor. Receptors containing β2- or β3-, but not β1 subunits, are highly sensitive to the agent. Here, chimeric β1/β2 subunits coexpressed in Xenopus laevis oocytes with human α6 and γ2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the β3 subunit to Ser (the homologous residue in β1), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the β1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk−) cells stably expressing the α6β3γ2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.

Thermus thermophilus: A link in evolution of the tRNA-dependent amino acid amidation pathways

Thermus thermophilus possesses an aspartyl-tRNA synthetase (AspRS2) able to aspartylate efficiently tRNAAsp and tRNAAsn. Aspartate mischarged on tRNAAsn then is converted into asparagine by an ω amidase that differs structurally from all known asparagine synthetases. However, aspartate is not misincorporated into proteins because the binding capacity of aminoacylated tRNAAsn to elongation factor Tu is only conferred by conversion of aspartate into asparagine. T. thermophilus additionally contains a second aspartyl-tRNA synthetase (AspRS1) able to aspartylate tRNAAsp and an asparaginyl-tRNA synthetase able to charge tRNAAsn with free asparagine, although the organism does not contain a tRNA-independent asparagine synthetase. In contrast to the duplicated pathway of tRNA asparaginylation, tRNA glutaminylation occurs in the thermophile via the usual pathway by using glutaminyl-tRNA synthetase and free glutamine synthesized by glutamine synthetase that is unique. T. thermophilus is able to ensure tRNA aminoacylation by alternative routes involving either the direct pathway or by conversion of amino acid mischarged on tRNA. These findings shed light on the interrelation between the tRNA-dependent and tRNA-independent pathways of amino acid amidation and on the processes involved in fidelity of the aminoacylation systems.

A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly

Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the “mutant ali-esterase hypothesis”). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 → Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp137 substitution alone is responsible for both the loss of E3’s carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp137 in the homologous position in acetylcholinesterase suggests that Asp137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.

Evidence that the 42- and 40-amino acid forms of amyloid β protein are generated from the β-amyloid precursor protein by different protease activities

Cerebral deposition of the amyloid β protein (Aβ) is an early and invariant feature of Alzheimer disease (AD). Whereas the 40-amino acid form of Aβ (Aβ40) accounts for ≈90% of all Aβ normally released from cells, it appears to contribute only to later phases of the pathology. In contrast, the longer more amyloidogenic 42-residue form (Aβ42), accounting for only ≈10% of secreted Aβ, is deposited in the earliest phase of AD and remains the major constituent of most amyloid plaques throughout the disease. Moreover, its levels have been shown to be increased in all known forms of early-onset familial AD. Thus, inhibition of Aβ42 production is a prime therapeutic goal. The same protease, γ-secretase, is assumed to generate the C termini of both Aβ40 and Aβ42. Herein, we analyze the effect of the compound MDL 28170, previously suggested to inhibit γ-secretase, on β-amyloid precursor protein processing. By immunoprecipitating conditioned medium of different cell lines with various Aβ40- and Aβ42-specific antibodies, we demonstrate a much stronger inhibition of the γ-secretase cleavage at residue 40 than of that at residue 42. These data suggest that different proteases generate the Aβ40 and Aβ42 C termini. Further, they raise the possibility of identifying compounds that do not interfere with general β-amyloid precursor protein metabolism, including Aβ40 production, but specifically block the generation of the pathogenic Aβ42 peptide.

Inorganic polyphosphate kinase is required to stimulate protein degradation and for adaptation to amino acid starvation in Escherichia coli

Inorganic polyphosphate (polyP) kinase was studied for its roles in physiological responses to nutritional deprivation in Escherichia coli. A mutant lacking polyP kinase exhibited an extended lag phase of growth, when shifted from a rich to a minimal medium (nutritional downshift). Supplementation of amino acids to the minimal medium abolished the extended growth lag of the mutant. Levels of the stringent response factor, guanosine 5′-diphosphate 3′-diphosphate, increased in response to the nutritional downshift, but, unlike in the wild type, the levels were sustained in the mutant. These results suggested that the mutant was impaired in the induction of amino acid biosynthetic enzymes. The expression of an amino acid biosynthetic gene, hisG, was examined by using a transcriptional lacZ fusion. Although the mutant did not express the fusion in response to the nutritional downshift, Northern blot analysis revealed a significant increase of hisG-lacZ mRNA. Amino acids generated by intracellular protein degradation are very important for the synthesis of enzymes at the onset of starvation. In the wild type, the rate of protein degradation increased in response to the nutritional downshift whereas it did not in the mutant. Supplementation of amino acids at low concentrations to the minimal medium enabled the mutant to express the hisG-lacZ fusion. Thus, the impaired regulation of protein degradation results in the adaptation defect, suggesting that polyP kinase is required to stimulate protein degradation.

Detection of protein fold similarity based on correlation of amino acid properties

An increasing number of proteins with weak sequence similarity have been found to assume similar three-dimensional fold and often have similar or related biochemical or biophysical functions. We propose a method for detecting the fold similarity between two proteins with low sequence similarity based on their amino acid properties alone. The method, the proximity correlation matrix (PCM) method, is built on the observation that the physical properties of neighboring amino acid residues in sequence at structurally equivalent positions of two proteins of similar fold are often correlated even when amino acid sequences are different. The hydrophobicity is shown to be the most strongly correlated property for all protein fold classes. The PCM method was tested on 420 proteins belonging to 64 different known folds, each having at least three proteins with little sequence similarity. The method was able to detect fold similarities for 40% of the 420 sequences. Compared with sequence comparison and several fold-recognition methods, the method demonstrates good performance in detecting fold similarities among the proteins with low sequence identity. Applied to the complete genome of Methanococcus jannaschii, the method recognized the folds for 22 hypothetical proteins.

Herbicide sensitivity determinant of wheat plastid acetyl-CoA carboxylase is located in a 400-amino acid fragment of the carboxyltransferase domain

A series of chimeral genes, consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) cDNA, and yeast ACC1 3′-tail, was used to complement a yeast ACC1 mutation. These genes encode a full-length plastid enzyme, with and without the putative chloroplast transit peptide, as well as five chimeric cytosolic/plastid proteins. Four of the genes, all containing at least half of the wheat cytosolic ACCase coding region at the 5′-end, complement the yeast mutation. Aryloxyphenoxypropionate and cyclohexanedione herbicides, at concentrations below 10 μM, inhibit the growth of haploid yeast strains that express two of the chimeric ACCases. This inhibition resembles the inhibition of wheat plastid ACCase observed in vitro and in vivo. The differential response to herbicides localizes the sensitivity determinant to the third quarter of the multidomain plastid ACCase. Sequence comparisons of different multidomain and multisubunit ACCases suggest that this region includes part of the carboxyltransferase domain, and therefore that the carboxyltransferase activity of ACCase (second half-reaction) is the target of the inhibitors. The highly sensitive yeast gene-replacement strains described here provide a convenient system to study herbicide interaction with the enzyme and a powerful screening system for new inhibitors.

A σ32 mutant with a single amino acid change in the highly conserved region 2.2 exhibits reduced core RNA polymerase affinity

σ32, the product of the rpoH gene in Escherichia coli, provides promoter specificity by interacting with core RNAP. Amino acid sequence alignment of σ32 with other sigma factors in the σ70 family has revealed regions of sequence homology. We have investigated the function of the most highly conserved region, 2.2, using purified products of various rpoH alleles. Core RNAP binding analysis by glycerol gradient sedimentation has revealed reduced core RNAP affinity for one of the mutant σ32 proteins, Q80R. This reduced core interaction is exacerbated in the presence of σ70, which competes with σ32 for binding of core RNAP. When a different but more conserved amino acid was introduced at this position by site-directed mutagenesis (Q80N), this mutant sigma factor still displayed a significant reduction in its core RNAP affinity. Based on these results, we conclude that at least one specific amino acid in region 2.2 is involved in core RNAP interaction.

Structure and function in rhodopsin: Rhodopsin mutants with a neutral amino acid at E134 have a partially activated conformation in the dark state*

The Glu-134–Arg-135 residues in rhodopsin, located near the cytoplasmic end of the C helix, are involved in G protein binding, or activation, or both. Furthermore, the charge-neutralizing mutation Glu-134 to Gln-134 produces hyperactivity in the activated state and produces constitutive activity in opsin. The Glu/Asp-Arg charge pair is highly conserved in equivalent positions in other G protein-coupled receptors. To investigate the structural consequences of charge-neutralizing mutations at Glu-134 and Arg-135 in rhodopsin, single spin-labeled side chains were introduced at sites in the cytoplasmic domains of helices C (140), E (227), F (250), or G (316) to serve as “molecular sensors” of the local helix bundle conformation. In each of the spin-labeled rhodopsins, a Gln substitution was introduced at either Glu-134 or Arg-135, and the electron paramagnetic resonance spectrum of the spin label was used to monitor the structural response of the helix bundle. The results indicate that a Gln substitution at Glu-134 induces a photoactivated conformation around helices C and G even in the dark state, an observation of potential relevance to the hyperactivity and constitutive activity of the mutant. In contrast, little change is induced in helix F, which has been shown to undergo a dominant motion upon photoactivation. This result implies that the multiple helix motions accompanying photoactivation are not strongly coupled and can be induced to take place independently. Gln substitution at Arg-135 produces only minor structural changes in the dark- or light-activated conformation, suggesting that this residue is not a determinant of structure in the regions investigated, although it may be functionally important.