Specific Amino Acid Composition Research Articles

Environment specific substitution tables for thermophilic proteins

BackgroundThermophilic organisms are able to live at high temperatures ranging from 50 to > 100°C. Their proteins must be sufficiently stable to function under these extreme conditions; however, the basis for thermostability remains elusive. Subtle differences between thermophilic and mesophilic molecules can be found when sequences or structures from homologous proteins are compared, but often these differences are family-specific and few general rules have been derived. The availability of complete genome sequences has now made it feasible to perform a large-scale comparison between mesophilic and thermophilic proteins, the latter of which primarily come from archaeal genomes although a few complete genomes of thermophilic eubacteria are also available.ResultsWe compared mesophilic proteins with their thermophilic counterparts of archaeal or eubacterial origins independently. This was based on the assumption that in these two kingdoms, different mechanisms may have been exploited for the adaptation of proteins at high temperatures. We derived the environment specific amino acid compositions of thermophilic proteins from 10 archaeal and seven eubacterial genomes, by aligning a large number of sequences from thermophilic proteins with their close mesophilic homologues of known three-dimensional (3D) structure. We further analysed environment specific substitutions, which lead from mesophilic proteins to either archaeal or eubacterial thermophilic proteins.ConclusionOur comparisons were based on homology-based structural predictions for a large number of thermophilic proteins. We demonstrated that thermal adaptation in the archaeal and eubacterial kingdoms is achieved in different ways. The main differences concern the usage of Gln, Ile and positively charged amino acids. In particular archaeal organisms appeared to have acquired thermostability by substituting non-charged polar amino acids (such as Gln) with Glu and Lys, and non-polar amino acids with Ile on the surface of proteins.

Use of Amino Acid Composition to Predict Ligand-Binding Sites

A novel method for predicting the binding sites for druglike compounds on the surface of proteins was developed on the basis of the specific amino acid composition observed at the ligand-binding sites of ligand-protein complexes determined by X-ray analysis. A profile representing the preference of each of the 20 standard amino acids at the binding sites of druglike molecules was obtained for a small set of high-quality complex structures. An index termed propensity for ligand binding (PLB) was created from these profiles. The PLB index was used to predict the propensity of binding for 804 ligands at all potential binding sites on the proteins whose structures were determined by X-ray analysis. If the sites with the first two highest PLB indices are taken into consideration, the successfully predicted sites reached a high percentage of 86. The PLB prediction is relatively simple, but the validation study showed that it is both fast and accurate to detect ligand-binding sites, especially the binding sites of druglike molecules. Therefore, the PLB index can be used to predict the ligand-binding sites of uncharacterized protein structures and also to identify novel drug-binding sites of known drug targets.

A Novel-type Substrate-selectivity Filter and ER-exit Determinants in the UapA Purine Transporter

We present a functional analysis of the last alpha-helical transmembrane segment (TMS12) of UapA, a uric acid-xanthine/H+ symporter in Aspergillus nidulans and member of the nucleobase-ascorbate transporter (NAT) family. First, we performed a systematic mutational analysis of residue F528, located in the middle of TMS12, which was known to be critical for UapA specificity. Substitution of F528 with non-aromatic amino acid residues (Ala, Thr, Ser, Gln, Asn) did not affect significantly the kinetics of UapA for its physiological substrates, but allowed high-capacity transport of several novel purines and pyrimidines. Allele-specific combinations of F528 substitutions with mutations in Q408, a residue involved in purine binding, led to an array of UapA molecules with different kinetic and specificity profiles. We propose that F528 plays the role of a novel-type selectivity filter, which, in conjunction with a distinct purine-binding site, control UapA-mediated substrate translocation. We further studied the role of TMS12 by analysing the effect of its precise deletion and chimeric molecules in which TMS12 was substituted with analogous domains from other NATs. The presence of any of the TMS12 tested was necessary for ER-exit while their specific amino acid composition affected the kinetics of chimeras.

Trophic-level interpretation based on d15N values: implications of tissue-specific fractionation and amino acid composition

Stable nitrogen isotope ratios are routinely used to disentangle trophic relationships. Several authors have discussed factors in addition to diet that might contribute to variability in δ 15 N of consumers, but few studies have explored such factors in detail. For a better understanding of tis- sue-specific differences in δ 15 N, we examined postlarval euphausiids across a variety of seasons and regions in the Southern Ocean. The concentration and δ 15 N of individual amino acids were analysed to account for both the biochemical and physiological underpinnings of the observed bulk δ 15 N. Euphausiids showed consistent d 15 N differences of 1 to 2 ‰ between the digestive gland and abdom- inal segment, and between reproductively active males and females. These differences in bulk δ 15 N were accompanied by variations in relative proportions of amino acids (up to 5 mol %) and their δ 15 N (up to 11‰). Aspartic acid and glutamic acid had the strongest influence on bulk δ 15 N, due to their high abundance and variable δ 15 N values. Differences in relative proportions and/or δ 15 N of glycine and alanine were also important for bulk δ 15 N values. Isotopic variations in amino acids between gen- der and tissues were explained by dominant internal processes such as protein synthesis or degrada- tion for energy supply, and by differences in amino acid pool sizes. Despite the offset in bulk δ 15 N between females and males, several lines of evidence suggested that their trophic levels were simi- lar. Thus, specific amino acid composition and metabolism may confound trophic level interpretations of bulk δ 15 N values. Micronekton are normally analyzed whole in isotopic studies, and we suggest that their analyses should be restricted to comparable tissues such as muscles.

Free amino acid composition and botanical origin of honey

The main amino acids found in 31 Spanish honeys of five different single botanical origins, were proline, phenylalanine, tyrosine and lysine, followed by arginine, glutamic acid, histidine and valine. Principal component analysis explained 64% of the variance with the first three PC variables, the lavender honeys being the only group well differentiated. Although the best grouped honeys were those from orange blossom, overlapping with eucalyptus honeys, on the one hand, and with the indistinguishable group formed by rosemary and thyme honeys, on the other hand, was observed. The Student–Newman–Keuls test allowed the grouping of rosemary, thyme and orange blossom honeys, whereas eucalyptus and lavender honeys showed specific amino acid compositions which made them different when compared with this group and also between themselves. Lavender honeys had the highest concentrations of tyrosine. The results obtained for the former honeys together with those obtained for another set of 17 samples, were used to establish a range for amino acid composition of Spanish honeys.

The Linker Region Plays an Important Role in the Interdomain Communication of the Response Regulator OmpR

OmpR is the response regulator of a two-component regulatory system that controls the expression of the porin genes ompF and ompC in Escherichia coli. This regulator consists of two domains joined by a flexible linker region. The amino-terminal domain is phosphorylated by the sensor kinase EnvZ, and the carboxyl-terminal domain binds DNA via a winged helix-turn-helix motif. In vitro studies have shown that amino-terminal phosphorylation enhances the DNA binding affinity of OmpR and, conversely, that DNA binding by the carboxyl terminus increases OmpR phosphorylation. In the present work, we demonstrate that the linker region contributes to this communication between the two domains of OmpR. Changing the specific amino acid composition of the linker alters OmpR function, as does increasing or decreasing its length. Three linker mutants give rise to an OmpF(+) OmpC(-) phenotype, but the defects are not due to a shared molecular mechanism. Currently, functional homology between response regulators is predicted based on similarities in the amino and carboxyl-terminal domains. The results presented here indicate that linker length and composition should also be considered. Furthermore, classification of response regulators in the same subfamily does not necessarily imply that they share a common response mechanism.

Origins of gene, genetic code, protein and life: comprehensive view of life systems from a GNC-SNS primitive genetic code hypothesis.

We have investigated the origin of genes, the genetic code, proteins and life using six indices (hydropathy, alpha-helix, beta-sheet and beta-turn formabilities, acidic amino acid content and basic amino acid content) necessary for appropriate three-dimensional structure formation of globular proteins. From the analysis of microbial genes, we have concluded that newly-born genes are products of nonstop frames (NSF) on antisense strands of microbial GC-rich genes [GC-NSF(a)] and from SNS repeating sequences [(SNS)n] similar to the GC-NSF(a) (S and N mean G or C and either of four bases, respectively). We have also proposed that the universal genetic code used by most organisms on the earth presently could be derived from a GNC-SNS primitive genetic code. We have further presented the [GADV]-protein world hypothesis of the origin of life as well as a hypothesis of protein production, suggesting that proteins were originally produced by random peptide formation of amino acids restricted in specific amino acid compositions termed as GNC-, SNS- and GC-NSF(a)-0th order structures of proteins. The [GADV]-protein world hypothesis is primarily derived from the GNC-primitive genetic code hypothesis. It is also expected that basic properties of extant genes and proteins could be revealed by considerations based on the scenario with four stages.

Principles governing amino acid composition of integral membrane proteins: application to topology prediction

A new method is suggested here for topology prediction of helical transmembrane proteins. The method is based on the hypothesis that the localizations of the transmembrane segments and the topology are determined by the difference in the amino acid distributions in various structural parts of these proteins rather than by specific amino acid compositions of these parts. A hidden Markov model with special architecture was developed to search transmembrane topology corresponding to the maximum likelihood among all the possible topologies of a given protein. The prediction accuracy was tested on 158 proteins and was found to be higher than that found using prediction methods already available. The method successfully predicted all the transmembrane segments in 143 proteins out of the 158, and for 135 of these proteins both the membrane spanning regions and the topologies were predicted correctly. The observed level of accuracy is a strong argument in favor of our hypothesis.

Ppc, the gene for phosphoenolpyruvate carboxylase from an extremely thermophilic bacterium, Rhodothermus obamensis: cloning, sequencing and overexpression in Escherichia coli.

The ppc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) of an extremely thermophilic bacterium, Rhodothermus obamensis, was directly sequenced by the thermal asymmetric interlaced (TAIL) PCR method. An ORF for a 937 amino acid polypeptide was found in the gene. The ppc gene had a high G+C content (66.2 mol%) and the third position of the codon exhibited strong preference for G or C usage (85.0 mol%). The calculated molecular mass was 107,848 Da, which was consistent with the molecular mass of the enzyme as determined by SDS-PAGE (100 kDa). The amino acid sequence of R. obamensis PEPC was closely related to that of PEPC from another thermophile, a Thermus sp., and from a mesophile, Corynebacterium glutamicum, exhibiting 45.3% or 37.7% identity and 61.5% or 56.5% similarity, respectively. By Southern analysis, the ppc gene was found to be present in a single copy in the genomic DNA of this organism. The cloned gene was expressed in Escherichia coli using a pET expression vector system and a thermostable recombinant PEPC was obtained. Comparison of the deduced amino acid sequences of the thermophilic and mesophilic PEPCs revealed distinct or common preferences for specific amino acid composition and substitutions in the two thermophilic enzymes.

Effect of germanium dioxide on growth ofSpirulina platensis

This study on the effect of different concentrations of germanium dioxide (GeO2) on the specific growth rate (SGR), pigment contents, protein content and amino acid composition ofSpirulina platensis showed that Ge was not the essential element of this alga; that GeO2 could speed up growth and raise protein content ofS. platensis, and could possibly influence the photosynthesis system. The concentration range of GeO2 beneficial to growth ofS. platensis is from 5–100mg/l. GeO2 is proposed to be utilized to remove contamination byChlorella spp. usually occurring in the cultivation ofSpirulina.

Allosteric Regulation in a Family of Enterobacterial Aspartate Transcarbamylases: Intramolecular Transmission of Regulatory Signals in Chimeric Enzymes

Several enterobacterial aspartate transcarbamylases (ATCases) exhibit a [2(c 3):3(r 2)] quaternary structure analogous to that of the Escherichia colienzyme. Despite their conserved quaternary structures, these enzymes present substantial differences in the co-operativity of substrate binding and in their allosteric regulation by nucleotide effectors. A comparison between different enzymatic species provides an opportunity to expand our understanding of the molecular basis of allostery in ATCase. Chimeric ATCases were constructed by exchanging subdomain regions involved in quaternary structural features, such as the r1-c4 regulatory-catalytic subunit interface analyzed in this study, in order to define the involvement of this interface in the several components of allosteric regulation. The r1-c4 interface was found to constitute an essential element for the recognition and the transmission of the ATP regulatory signal in the Serratia marcescensand the Proteus vulgarisATCases, as it does in the E. coliATCase. Besides, the specific amino acid composition of the C-terminal region of the regulatory chain and its interactions with the amino acid residues in the 240s loop of the catalytic chain (r1-c4 interactions) were found to modulate the amplitude of the enzyme's response to ATP. The C-terminal region of the regulatory chain did not appear to participate directly in the regulation of the three native ATCases by CTP. Even when CTP acts as an activator, as in the P. vulgarisand S. marcescensATCases, its signal follows a route distinct from that of the general activator ATP. Synergistic inhibition by CTP and UTP was found to involve the transmission of a specific UTP signal. This signal appeared different in the various ATCases, involving the C-terminal region of the regulatory chain in the E. coliand S. marcescensATCases but not in the P. vulgarisATCase.

Isolation and purification of amyloglucosidase from Halobacterium sodomense.

Amyloglucosidase from Halobacterium sodomense was purified by a combination of hydrophobic interaction chromatography and immobilized metal ion affinity chromatography at analytical and preparative scale with 75% recovery. The enzyme was found to be a dimer of two different subunits with molecular weights of 72,000 and 82,000 D, respectively, combining in a 175,000 D native protein. The specific activity, KM, and amino acid composition of the enzyme was determined.

Branched-chain amino acids augment neither albuminuria nor albumin synthesis in nephrotic rats

Both albuminuria (UalbV) and albumin synthesis (AlbSyn) are modulated by dietary protein in nephrotic rats, but the agent(s) linking diet to altered UalbV and AlbSyn is unknown. Others have reported that branched-chain amino acids (BCAA) cause neither increased renal blood flow nor glomerular filtration rate (GFR) normally induced by dietary protein nor increased blood glucagon thought to be necessary for protein-mediated effects on renal hemodynamics. The effect of BCAA on UalbV is unknown. Because BCAA increase AlbSyn in tissue culture and after a fast, it is possible that feeding BCAA may increase AlbSyn but not UalbV in nephrosis. Nephrotic rats were fed either 8.5% casein (LP); 21% casein (NP); 8.5% casein supplemented with valine, leucine, and isoleucine to the total amount provided by a 21% casein diet (2.37%) (LBC); or 8.5% casein plus 12.5% BCAA providing a diet isonitrogenous to 21% casein (HBC). UalbV and AlbSyn were significantly greater in NP compared with LP, LBC, or HBC and were the same in the latter three groups. Glucagon was infused into nephrotic rats fed 8.5% casein either subcutaneously or intraperitoneally in quantities sufficient to increase plasma levels to over 10 times control but had no effect on UalbV. The ability of dietary protein to increase AlbSyn or UalbV is not a result of total alpha-amino nitrogen intake but is a result of the specific amino acid composition of the diet and must result entirely from the effect of one or more non-BCAA. Increased blood glucagon alone has no effect on UalbV.

Amino acid composition of aragonitic concholin in the shell of Arctica islandica

Haugen, J. E. & Sejrup, H. P. 1990 04 15: Amino acid composition of aragonitic conchiolin in the shell of Arctica islandica. Lethaia, Vol. 23, pp. 133–141. Oslo. ISSN 0024–1164. The distribution of amino acids within the two aragonitic shell layers of modern specimens of the mollusc Arctica islandica (Linne) has been studied in detail. The mean total hydrolyzed amino acid content was 19 nmol*mg in the inner layer and 15 nmol/mg in the outer layer. No significant difference in amino acid composition could be found between the two layers. The layers contained minor amounts of free amino acids which made up 0.3–0.7% of the total hydrolyzed amino acid content. The composition of the free amino acid fraction was very similar in each of the two layers, but differed somewhat from the total hydrolyzed fraction. The total hydrolyzed fraction was dominated by aspartic acid, glycine. alanine and glutamic acid, which together made up 62% of the total amount of amino acids, whereas the free fraction was dominated by Asp. Ser, Gly and Tyr. Amino acid content and composition within a single layer showed little variation from umbo to the ventral margin. The amino acid composition is in accordance with previously reported data on similar mineral structures which support the theory of structure specific rather than species specific amino acid composition. * Arctica islandica. amino acids. shell structure.

Cloning and sequence analysis of the arginine deiminase gene from Mycoplasma arginini

Arginine deiminase from Mycoplasma arginini was purified. The purified enzyme has a molecular weight of 46,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Its specific activity (20 units/mg protein) and amino acid composition showed a strong similarity to that of the Mycoplasma arthritidis arginine deiminase. The amino acid sequences of the N-terminal region and three internal peptides generated by enzymatic cleavage of the purified protein were determined. Using a synthetic oligonucleotide mixture complementary to part of the determined N-terminal amino acid sequence, the gene coding for arginine deiminase was isolated from a phage library. A nucleotide sequence of 2189 bp encoding the gene was determined. An open reading frame (ORF) contained the amino acid sequences corresponding to the determined N-terminal region and the three internal peptides of arginine deiminase. Thus it was concluded that this ORF encoded the arginine deiminase, a 385 amino acid polypeptide (mol.wt. 43,900 daltons). The three tryptophan residues in the sequenced peptides align with UGA codons in the nucleotide sequence, indicating that the nonsense codon UGA is used as a tryptophan codon in M. arginini.

The composition of the proteinaceous gums exuded by some Leucaena species, subspecies and hybrids

Analytical data are presented for the sugar and amino acid compositions of the gum exudates from Leucaena leucocephala (four specimens), L.pallida, L.leucocephala × L.pallida (two specimens) and L.lanceolata subsp. sousae (two specimens). Conversion factors have been calculated from the amino acid data to facilitate the conversion of Kjeldahl nitrogen values to protein contents; the factors are considerably higher than that assumed in an earlier study of Leucaena gum. The data confirm and extend the previous indication that Leucaena gum has a strongly negative specific rotation, with sugar and amino acid compositions that are similar to those of commercial gum arabic [ Acacia Senegal (L.) Willd.]. Commercial possibilities and mechanisms of gum formation are discussed.

Phenylalanine dehydrogenase of Bacillus badius. Purification, characterization and gene cloning.

Phenylalanine dehydrogenase produced by Bacillus badius IAM 11059 was purified from the crude extract of B. badius to homogeneity, as judged by disc gel electrophoresis. The enzyme has an isoelectric point of 3.5 and a relative molecular mass, Mr, of 310,000-360,000. The enzyme is composed of identical subunits with an Mr 41,000-42,000. The substrate specificity of the enzyme in the oxidative deamination reaction was high for L-phenylalanine, but rather low in the reductive amination reaction, with phenylpyruvate, p-hydroxyphenylpyruvate, and 2-oxohexanoate. The gene for the enzyme was cloned into Escherichia coli with plasmid pBR322 as a vector. The enzyme was expressed in high level in E. coli. The enzyme produced by E. coli transformant was purified to homogeneity and shown to be identical to that of B. badius IAM 11,059 with respect to the specific activity, Mr, subunit structure and amino acid composition.

Effect of individual amino acids on plasma cholesterol level in rats fed a high cholesterol diet.

The effect of individual amino acids added to a cholesterol containing casein diet on the plasma cholesterol level of rats was systematically investigated with objective of confirming the possibility that specific amino acids are responsible for the regulation of plasma cholesterol level by dietary proteins. Half the amino acids tested were effective to some degree in influencing the plasma cholesterol level when amino acids were added singly to the diet at a 5% level. However, only sulfur containing amino acids had significant effects when added to the diet at a 1% level; Met enhanced and Cys reduced the plasma cholesterol level. In addition, the results of an experiment with amino acid mixture diets with different Met, Cys, and/or Gly contents indicated the importance of sulfur amino acids and also of Gly in plasma cholesterol regulation. The results obtained here support the notion that the plasma cholesterol level of rats can be influenced by specific amino acids or amino acid compositions of dietary proteins, at least under the experimental conditions used.

Effect of Environmental Salinity on the Free Amino Acid Composition and Concentration in Penaeid Shrimp

ABSTRACTWhite shrimp, Penaeus vannamei, kept in laboratory aquaria at a salinity of 35 parts per thousand (ppt) were subjected to increased and decreased salinity changes by either 5, 15 or 25 ppt. Shrimp were periodically removed from the aquaria and analyzed for total free amino acid nitrogen and also specific free amino acid composition. Decreasing salinities resulted in corresponding decreases in free amino acid concentration; minimum values were reached after 24 ‐ 48 hr. Increasing salinities were likewise shown to result in corresponding increases in the tissue free amino acid concentration with maximum values being reachedin 24 hr. Glycine, proline, arginine, serine/threonine, and alanine were the most significant contributors, comprising from 93 ‐ 96% of the total free amino acid pool.

Hormone-induced rearrangement of locust haemolymph lipoproteins: The involvement of glycoprotein C 2

Formation of lipoprotein A + and elevation of lipoprotein fraction O in locust ( Locusta migratoria migratorioides) haemolymph as induced by adipokinetic hormone (AKH) includes the participation of non-lipid carrying proteins (fraction C), which was examined in more detail. By using gel filtration chromatography, the rather heterogenous C-proteins were resolved into three protein fractions, only one of which (C 2) appeared to be actually involved in the lipoprotein reassociation. The changes in amino acid composition of the elevated lipoprotein fractions as compared with those from the lipoproteins in the resting situation are accounted for by the contribution of the rather specific amino acid composition of this C 2-fraction. Polyacrylamide gel electrophoresis (PAGE) indicates that the C 2-protein is migrating as only one band; SDS-PAGE revealed that the C 2-protein consists of one single polypeptide chain with an approximate molecular weight of 20,000. This chain is also recovered in the subunit structure of the lipoprotein fractions induced by AKH-injection (A +, O AKH) in contrast with that of the lipoprotein fractions in resting haemolymph. Unlike the other C-proteins, protein C 2 displayed immunoreactivity with antiserum raised against lipoprotein A +. From carbohydrate analyses, C 2 appeared to be a glycoprotein containing approx. 12.5% carbohydrate. In vivo pilot studies on the dynamics of C 2-proteins using 3H-labelled glycoprotein C 2 gave evidence for the incorporation of radiolabel into both A + and O AKH. Possible functions of the involvement of the glycoprotein to A + formation are discussed.