Search for structural factors responsible for the stability of proteins from thermophilic organisms

Database including 392 homologous pairs of proteins from thermophilic and mesophilic organisms was created. Using this database we have found that proteins from termophilic organisms contain more atom-atom contacts per residue in comparison with mesophilic homologues. Contribution to increase of the number of contacts gives exterior amino acid residues, accessible for the solvent. Amino acid composition of interior, inaccessible for the solvent, and exterior amino acid residues of proteins from thermophilic and mesophilic organisms were analyzed. We have obtained that exterior residues of proteins from thermophilic organisms contain more such amino acid residues as Lys, Arg and Glu and smaller such amino acid residues as Ala, Asp, Asn. Gln, Ser, and Thr in comparison with proteins from mesophilic organisms. Amino acid compositions of interior residues of considered proteins are not different.     

Unorthodox expression of an enzyme: evidence for an untranslated region within carA from Pseudomonas aeruginosa.

The genes encoding carbamoylphosphate synthetase from Pseudomonas aeruginosa PAO1 were cloned in Escherichia coli. Deletion and transposition analysis determined the locations of carA, encoding the small subunit, and carB, encoding the large subunit, on the chromosomal insert. The nucleotide sequence of carA and the flanking regions was determined. The derived amino acid sequence for the small subunit of carbamoylphosphate synthetase from P. aeruginosa exhibited 68% homology with its counterparts in E. coli and Salmonella typhimurium. The derived sequences in the three organisms were essentially identical in the three polypeptide segments that are conserved in glutamine amidotransferases but showed low homology at the amino- and carboxy-terminal regions. The amino-terminal amino acid sequences were determined for the large and small subunits. The first 15 amino acids of the large subunit were identical to those derived from the carB sequence. However, comparison of the derived sequence for carA with the amino-terminal amino acid sequence for the small subunit suggested that codons 5 to 8 are not translated. The DNA sequence for the region encompassing these four codons was confirmed by direct sequencing of chromosomal DNA after amplification by the polymerase chain reaction. The mRNA sequence was also deduced by in vitro synthesis of cDNA, enzymatic amplification, and sequencing, confirming that 12 nucleotides in the 5' terminal of carA are transcribed but are not translated.     

Similarities in protein amino acid composition in connection with principles of protein evolution

The amino acid composition of human alcohol dehydrogenase (ADH) was compared with alcohol dehydrogenases from different organisms and with other proteins. Similar amino acid sequences in human ADH (template protein) and in other proteins were determined by means of an original computer program. Analysis of amino acid motifs reveals that the ADHs from evolutionary more close organisms have more common amino acid sequences. The quantity measure of amino acid similarity was the number of similar motifs in analyzed protein per protein length. This value was measured for ADHs and for different proteins. For ADHs, this quotient was higher than for proteins with different functions; for vertebrates it correlated with evolutionary closeness. The similar operation of motif comparison was made with the help of program complex “MEME”. The analysis of ADHs revealed 4 motifs common to 6 of 10 tested organisms and no such motifs for proteins of different function. The conclusion is that general amino composition is more important for protein function than amino acid order and for enzymes of similar function it better correlates with evolutionary distance between organisms.     

Evidence for cysteine clustering in thermophilic proteomes

Through linguistic analysis, we show that the presence of an amino acid at a given position within a proteome positively influences the presence of identical amino acids at nearby positions. We call this phenomenon 'amino acid clustering'. Clustering extends well beyond the closest neighbouring sites and is particularly pronounced for cysteine and tryptophan. Cysteine clusters preferentially form CXXC structures, and they are often involved in metal coordination or disulfide bond formation. Cysteine clustering shows a clear correlation with the growth temperature of the organism. This seems to be a general property of living organisms.     

Isolation and Amino Acid Composition of Ribosomal Proteins from Bacillus stearothermophilus

Twelve of the proteins from the 30S ribosome of Bacillus stearothermophilus were isolated by preparative disc electrophoresis. Amino acid analyses of these proteins showed them to be different from each other. The gross amino acid composition of 30S ribosomal protein from B. stearothermophilus and Escherichia coli are virtually identical. A number of the proteins of B. stearothermophilus had electrophoretic mobilities similar or identical to 30S ribosomal proteins of E. coli. However, there was little similarity between the two organisms in amino acid composition of individual proteins. There were no unusual chemical features of the B. stearothermophilus proteins which could explain the relative thermal stability of this organism's ribosomes.     

Occurrence of six-amino-acid motifs in three eukaryotic proteomes

Currently 18 hereditary neurological diseases are known to be associated with such mutations as multiple insertions of a single amino acid into the protein sequence. Therefore, investigation of the functional purpose of simple amino acid motifs becomes an important biological task. In this work, we studied the frequencies of motifs consisting of six identical amino acids and of simple six-amino-acid motifs consisting of two randomly located amino acids. The investigation was conducted on three eukaryotic proteomes of the well-studied model organisms, Homo sapiens, Drosophila melanogaster, and Caenorhabditis elegans. We showed that many simple motifs occurred very frequently; the data on the frequency were presented at These results suggest such motifs to be responsible for common functions of non-homologous and unrelated proteins in different organisms.     

N5-(1-carboxyethyl)-ornithine, a new amino acid from the intracellular pool of Streptococcus lactis.

Intracellular concentrations of amino acids were determined in cells of Streptococcus lactis 133 during growth in complex, spent, and chemically defined media. Glutamic and aspartic acids represented the major constituents of the amino acid pool. However, organisms grown in spent medium or in defined medium supplemented with ornithine also contained unusually high levels of two additional amino acids. One of these amino acids was ornithine. The second compound exhibited properties of a neutral amino acid by coelution with valine from the amino acid analyzer. The compound did not, however, comigrate with valine or any other standard amino acid by two-dimensional thin-layer chromatography. The unknown amino acid was purified by paper and thin-layer chromatography, and its molecular structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy. This new amino acid was shown to be N5-(1-carboxyethyl)-ornithine. The 14C-labeled compound was formed by cells of S. lactis 133 during growth in spent medium or defined medium containing [14C]ornithine. Formation of the derivative by resting cells required ornithine and the presence of a metabolizable sugar. N5-(1-Carboxyethyl)-ornithine was synthesized chemically from both poly-S-ornithine and (2S)-N2-carbobenzyloxy-ornithine as a 1:1 mixture of two diastereomers. The physical and chemical properties of the amino acid purified from S. lactis 133 were identical to those of one of the synthetic diastereomers. The bis-N-trifluoroacetyl-di-n-butyl esters of the natural and synthetic compounds generated identical gas chromatography-mass spectrometry spectra. A mechanism is suggested for the in vivo synthesis of N5-(1-carboxyethyl)-ornithine, and the possible functions of this new amino acid are discussed.     

Selection on Synthesis Cost Affects Interprotein Amino Acid Usage in All Three Domains of Life

Most investigations of the forces shaping protein evolution have focussed on protein function. However, cells are typically 50%–75% protein by dry weight, with protein expression levels distributed over five orders of magnitude. Cells may, therefore, be under considerable selection pressure to incorporate amino acids that are cheap to synthesize into proteins that are highly expressed. Such selection pressure has been demonstrated to alter amino acid usage in a few organisms, but whether “cost selection” is a general phenomenon remains unknown. One reason for this is that reliable protein expression level data is not available for most organisms. Accordingly, I have developed a new method for detecting cost selection. This method depends solely on interprotein gradients in amino acid usage. Applying it to an analysis of 43 whole genomes from all three domains of life, I show that selection on the synthesis cost of amino acids is a pervasive force in shaping the composition of proteins. Moreover, some amino acids have different price tags for different organisms—the cost of amino acids is changed for organisms living in hydrothermal vents compared with those living at the sea surface or for organisms that have difficulty acquiring elements such as nitrogen compared with those that do not—so I also investigated whether differences between organisms in amino acid usage might reflect differences in synthesis or acquisition costs. The results suggest that organisms evolve to alter amino acid usage in response to environmental conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Hector Musto]     

Evolutionary changes reflected by the cellular amino acid composition

Summary Comparison of the amino acid composition of cell-proteins using 17 amino acids has been used to investigate the biological evolution of organisms such as bacteria, blue-green alga, green alga, fungi, slime mold, protozoa and vertebrates. The degree of difference in the amino acid ratios between any two groups reflects the degree of divergency in biological evolution. The amino acid composition of the Gram-negative bacteria (Escherichia coli,Klebsiella,Proteus, andVibrio alginolyticus) was identical. However, the amino acid composition ofStaphylococcus aureus andBacillus subtilis, which are Gram-positive bacteria, differed from each other and from the Gram-negative bacteria. The amino acid composition of the blue-green alga (Cyanobacterium,Chroococidiopsis) was quite similar to that ofE. coli. A marked difference in the amino acid composition was observed betweenE. coli and green alga (Chlorella), and significant differences were observed betweenE. coli and other organisms, such as fungi, protozoa (Tetrahymena), slime mold (Dictyostelium discoideum) and vertebrates. In conclusion, the change in cellular amino acid composition reflects the divergence which has occurred during biological evolution, whereas a basic pattern of amino acid composition is maintained in spite of a long period of evolutional divergence among the various organisms. Thus, it is proposed that the primitive life forms established at the end of prebiotic evolution had a similar amino acid composition.     

A search for structural factors responsible for the stability of proteins from thermophilic organisms

A database was designed to include 392 pairs of homologous proteins from thermophilic and mesophilic organisms. Proteins from thermophilic organisms proved to contain more atom-atom contacts per residue as compared with their mesophilic homologs. Solvent-accessible exterior amino acid residues contribute to the increase in the number of contacts. The amino acid composition was analyzed for internal (solvent-inaccessible) and exterior amino acid residues of thermophilic and mesophilic proteins. The exterior residues of thermophils have higher contents of Lys, Arg, and Glu and lower contents of Ala, Asp, Asn, Gln, Ser, and Thr as compared with mesophilic proteins. Interior protein regions did not differ in amino acid composition.     

Cloning and nucleotide sequence of braC, the structural gene for the leucine-, isoleucine-, and valine-binding protein of Pseudomonas aeruginosa PAO.

The gene for the leucine-, isoleucine-, and valine-binding protein (LIVAT-BP) in Pseudomonas aeruginosa PAO was isolated, and its nucleotide sequence was determined. The gene consisted of 1,119 nucleotides specifying a protein of 373 amino acid residues. Determination of the N-terminal amino acid sequence of the LIVAT-BP purified from P. aeruginosa shock fluid suggested that the N-terminal 26 residues of the gene product are cleaved off posttranslationally, showing the characteristic features of procaryotic signal peptides. The amino acid composition of the mature product predicted from the nucleotide sequence was in good agreement with that of the purified LIVAT-BP. The plasmid carrying the LIVAT-BP gene restored the activity of the high-affinity branched-chain amino acid transport system (the leucine, isoleucine, valine [LIV-I] transport system) in the braC310 mutant of P. aeruginosa, confirming that braC is the structural gene for LIVAT-BP. The mutant LIVAT-BP lacking a 16-amino-acid peptide in the middle was found to be functional in the LIV-I transport system. LIVAT-BP showed extensive homology (51% identical) to the LIV- and leucine-specific-binding proteins of Escherichia coli K-12, which are coded for by the livJ and livK genes, respectively, suggesting that the role of the proteins in the LIV-I transport systems is analogous in both organisms.     

Comparative studies of S-layer proteins from Bacillus stearothermophilus strains expressed during growth in continuous culture under oxygen-limited and non-oxygen-limited conditions.

The specific properties of S-layer proteins from three different Bacillus stearothermophilus strains revealing oblique, square, or hexagonal lattice symmetry were preserved during growth in continuous culture on complex medium only under oxygen-limited conditions in which glucose was used as the sole carbon source. When oxygen limitation was relieved, amino acids became metabolized, cell density increased, and different S-layer proteins from wild-type strains became rapidly replaced by a new common type of S-layer protein with an apparent subunit molecular weight of 97,000 which assembled into an identical oblique (p2) lattice type. During switching from wild-type strains to variants, patches of the S-layer lattices characteristics for wild-type strains, granular regions, and areas with oblique lattice symmetry could be observed on the surface of individual cells from all organisms. The granular regions apparently consisted of mixtures of the S-layer proteins from the wild-type strains and the newly synthesized p2 S-layer proteins from the variants. S-layer proteins from wild-type strains possessed identical N-terminal regions but led to quite different cleavage products upon peptide mapping, indicating that they are encoded by different genes. Chemical analysis including N-terminal sequencing and peptide mapping showed that the oblique S-layer lattices synthesized under increased oxygen supply were composed of identical protein species.     

Spare parts for helix-helix interaction.

About 6000 contact regions (patches) of helix-to-helix packing from 300 well-resolved non-homologous protein structures were considered. The patches were defined by the spatial helical neighbors and were estimated in atomic detail using a variable distance criterion. The following questions are addressed. (1) Are the amino acid preferences and atomic composition of distinct types of helical patches indicative for the type of their neighbor? Distributions of size, atomic composition and packing density are compared for different types of helical interfaces. Thereby contact preferences are derived for parts of secondary structures adjoining each other or pointing towards the solvent. (2) Is it possible to cluster helical patches according to their structural similarity? For these purposes the patches were classified with an automatic sequence-independent superposition procedure which yields a distinctively reduced set of representative interfaces. On this basis, the methodology for finding exchangeable patches in different proteins is demonstrated.     

On the trends in protein molecular evolution: Amino acid composition

Data on the amino acid composition of proteins having various functions from organisms representing different evolutionary levels (83 superfamilies) are used in order to elucidate the trends in protein molecular evolution. The interconnections evolutionary rate (rate of mutation acceptance) — amino acid composition, and evolutionary level of the organism — amino acid composition (in case of proteins of the same or very similar function) are studied. The amino acid compositions of proteins performing jointly an evolutionarily old functions are also juxtaposed. The mean contemporary protein composition is used as a basis for comparison. The obtained results are evidence in favour of the existence of a trend for an increase of the special amino acids (Met, Ile, Gln, His, Lys, Asn, Phe, Tyr, Trp, Cys) at the expense of the usual ones (Thr, Pro, Ala, Ser, Arg, Gly, Leu, Val, Glu, Asp). The tests of statistical significance of the obtained results (comparison of the mean compositions of proteins from low evolutionary level organisms with that of all sequenced proteins; comparison of the mean contemporary protein composition with that obtained after simulation of the evolutionary process) confirm and universalize the observed trend. The above results direct the attention to the concept of a smaller number of amino acids in the ancient proteins and respectively simpler genetic code. A fluctuation around the initial primitive level is suggested to explain the conservatism of proteins of the same function in evolutionarily low level organisms. The observed trend could be applied for designing new proteins.     

Beta-subunit of ATP-synthase: a useful marker for studying the phylogenetic relationship of eubacteria

The genes encoding the beta-subunits of ATP-synthases (ATPases) from Bacteroides fragilis DSM 2151, Cytophaga lytica DSM 2039 and 'Taxeobacter ocellatus' were cloned. The nucleotide sequences were determined completely for the genes of the first two organisms and to a major part for that of 'T. ocellatus'. The predicted amino acid sequences were compared with previously published amino acid sequences of beta-subunits. Two characteristic insertions were found in genes from organisms belonging to the so-called bacteroides-cytophaga-flavo-bacterium group. The remaining structure shows a high degree of sequence similarity within this group. These data support the conclusions drawn from comparative 16S rRNA sequence analyses that organisms in this phenotypically heterogeneous group are phylogenetically related. A phylogenetic tree was constructed based on a distance matrix of optimally aligned amino acid sequences of beta-subunits of ATPases of various eubacteria, chloroplasts and mitochondria. It is in good agreement with a tree derived from 16S rRNA sequence analyses.     

Analysis of differences in amino acid substitution patterns, using multilevel G-tests

In this paper, a new algorithm is presented, which makes possible multilevel comparison of BLOSUM protein substitution matrices based on data from different groups of organisms. As an example, a comparison between substitution matrices based on data from two groups of bacterial genomes with different GC content is presented. Our approach includes evaluating the number of amino acid pairs in BLOCKS databases created separately for the two groups of bacteria using protein sequences deposited in the COG database. Differences of distributions of amino acid pair counts are tested using the chi-squared based G-test. Different analysis levels make it possible to distinguish different patterns of amino acid substitution. Application of the algorithm reveals statistically significant differences in amino acid substitution patterns between AT-rich and GC-rich groups of bacterial organisms. The differences are particularly visible in the overall substitution pattern, amino acid conservation pattern and in comparison of substitution patterns for single amino acids. The algorithm presented in this paper can be considered a novel method for multi-level comparison of amino acid substitution patterns. The presented approach is not limited to bacterial organisms and BLOSUM substitution matrices. Statistically significant differences between substitution patterns in the two groups of bacterial organisms with respect to amino acid conservation pattern can be the evidence of different rate of evolutionary change between AT-rich and GC-rich bacterial organisms.     

Primary structure deduced from complementary DNA sequence and expression in cultured cells of mammalian 4-hydroxyphenylpyruvic acid dioxygenase. Evidence that the enzyme is a homodimer of identical subunits homologous to rat liver-specific alloantigen F.

4-Hydroxyphenylpyruvic acid dioxygenase is an important enzyme in tyrosine catabolism in most organisms. From porcine and human liver cDNA libraries we isolated complementary DNA inserts for the enzyme. Protein sequence analysis of the porcine enzyme revealed a block of the amino terminus of the mature enzyme. Comparison of the amino acid sequence determined by Edman degradation of peptides derived from porcine liver 4-hydroxyphenylpyruvic acid dioxygenase with the nucleotide sequences revealed the primary structure of the porcine and human enzymes. The mature human and porcine enzymes have an 89% amino acid sequence identity in amino acid residues and are composed of 392 amino acid residues. A computer-assisted homology search revealed that the enzyme is 88% identical in amino acid sequence to rat liver-specific alloantigen F. A monoclonal antibody (mob 51), which can immunoprecipitate both the human and porcine enzymes, was developed. Cultured BMT-10 cells transfected with the cDNA insert of the human enzyme, using the expression vector pCAGGSneodE, produced a polypeptide with an M(r) of 43,000, which was immunoprecipitated with mob 51. Enzymic activity of the enzyme was detected in the transfected cells but not in the mock transfected cells. These findings suggest that the human 4-hydroxyphenylpyruvic acid dioxygenase is a homodimer of two identical subunits with an M(r) of 43,000. Liver-specific alloantigen F seems to be closely related to the enzyme or possibly to the subunit of the enzyme itself. Elucidation of the complete amino acid sequence of the enzyme is expected to reveal structure-function relationships of this metabolically important enzyme and to shed light on inherited disorders related to tyrosine metabolism, especially tyrosinemia types 1 and 3.     

NgUNC-119, Naegleria homologue of UNC-119, localizes to the flagellar rootlet

The UNC-119 family of proteins is ubiquitous in animals. The expression of UNC-119 is prominent in neural tissues including photoreceptor cells. Homologues of UNC-119 are also found in ciliated (or flagellated) single-celled organisms; however, the cellular distribution of this protein in protists is unknown. We cloned and characterized a homologue of unc-119 from the ameboflagellate Naegleria gruberi (Ngunc-119) and identified the cellular distribution of the protein. The Ngunc-119 open reading frame contained 570 nucleotides encoding a protein of 189 amino acids with a predicted molecular weight of 22.1 kDa, which is similar to that of Paramecium UNC-119 and Trypanosoma UNC-119. These three proteins are 46-48% identical in their amino acid sequences. The smaller NgUNC-119 corresponds to the conserved C-terminal 3/4 of the UNC-119 from multi-cellular organisms. The amino acid sequence of NgUNC-119 is 43-50% identical to that of the conserved C-terminal regions. NgUNC-119 was not found in growing amoebae but accumulated rapidly after the initiation of differentiation into flagellates. Indirect immunofluorescence staining of differentiating N. gruberi showed that NgUNC-119 begins to concentrate at a spot near the nucleus of differentiating cells and then elongates into a filamentous structure. Purification and indirect immunofluorescence staining of the Naegleria flagellar rootlet suggested that NgUNC-119 is a component of the flagellar rootlet.