Evolution of amino acid frequencies in proteins over deep time: inferred order of introduction of amino acids into the genetic code

To understand more fully how amino acid composition of proteins has changed over the course of evolution, a method has been developed for estimating the composition of proteins in an ancestral genome. Estimates are based upon the composition of conserved residues in descendant sequences and empirical knowledge of the relative probability of conservation of various amino acids. Simulations are used to model and correct for errors in the estimates. The method was used to infer the amino acid composition of a large protein set in the Last Universal Ancestor (LUA) of all extant species. Relative to the modern protein set, LUA proteins were found to be generally richer in those amino acids that are believed to have been most abundant in the prebiotic environment and poorer in those amino acids that are believed to have been unavailable or scarce. It is proposed that the inferred amino acid composition of proteins in the LUA probably reflects historical events in the establishment of the genetic code.     

编码序列的（G＋C）%与蛋白质的耐热性相关性分析

运用计算机统计方法,对以木糖异构酶为主的几个蛋白质家族的核酸和氨基酸序列进行分析,发现密码子各位上的（Ｇ＋Ｃ）％与编码序列的（Ｇ＋Ｃ）％成线性正相关,大多数氨基酸的含量与编码序列的（Ｇ＋Ｃ）％也存在相关性,按其相关性,将氨基酸分为正相关,负相关和不相关３类,对木糖异构酶氨基酸序列和酶的耐热性的统计发现,那些在统计学上显著的,可能提高蛋白质耐热性的氨基酸替换,往往伴随关编码序列中ＧＣ含量的上升,这提     

Investigation of periodic distributions of amino acids in the sequences of fiber proteins of bacteriophage T4

Sequences of amino acids of some fiber proteins may have a periodic structure. To analyze this periodicity Fourier transform of a mathematical image of symbolic sequence of amino acids in a protein is sometimes used. In this work we employed one (out of few possible) particular way of doing Fourier transform as the most straightforward and optimal. Employing this optimal Fourier transform method we analyzed periodicity of fiber proteins in bacteriophage T4. As a result we managed to confirm that a certain periodicity exists in the investigated proteins. It was found that for a number of proteins the alternation of elements of the same group in the amino acid sequence with a rather small period T = 15 exists, whereas for some other proteins alternations have small periods 10 and 8. The new result is a discovery of relatively large periods of amino acids alternations, which divide the amino acids sequence of the protein into 4 or 6 equal parts. These data on the amino acids periodicity allowed us to align amino acids sequences in accordance with the established periods of both types, in agreement with certain results obtained in X-ray crystallography and electron microscopy experiments.     

嗜热与嗜常温微生物的蛋白质氨基酸组成比较

嗜热微生物的嗜热特性与其蛋白质的高度热稳定性紧密相关。为了探索嗜热蛋白质的热稳定机制,比较嗜热和嗜常温微生物的蛋白质在氨基酸组成上的差别,收集110对分别来自嗜热和嗜常温微生物的同源蛋白质序列,比较两组蛋白质各种氨基酸含量以及疏水性氨基酸组成、疏水性指数和荷电氨基酸组成的差别,结果两者在多种氨基酸含量上存在微小但统计学上显著的差别,嗜热蛋白质比嗜常温蛋白质具有较高的平均疏水性和荷电氨基酸组成。对两组蛋白质的“脂肪族氨基酸指数”进行分析,证明嗜热蛋白质之所以具有较高的脂肪族氨基酸指数是由于其亮氨酸含量较高,与影响该指数的其它几种氨基酸无关;从而认为该指数的意义值得怀疑。通过对大量同源嗜热蛋白质和嗜常温蛋白质氨基酸组成的比较,能够揭示一些有关蛋白质热稳定性的普遍规律。     

Low Contents of Carbon and Nitrogen in Highly Abundant Proteins: Evidence of Selection for the Economy of Atomic Composition

Proteins that assimilate particular elements were found to avoid using amino acids containing the element, which indicates that the metabolic constraints of amino acids may influence the evolution of proteins. We suspected that low contents of carbon, nitrogen, and sulfur may also be selected for economy in highly abundant proteins that consume large amounts of the resources of cells. By analyzing recently available proteomic data in Escherichia coli, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, we found that at least the carbon and nitrogen contents in amino acid side chains are negatively correlated with protein abundance. An amino acid with a high number of carbon atoms in its side chain generally requires relatively more energy for its synthesis. Thus, it may be selected against in highly abundant proteins either because of economy in building blocks or because of economy in energy. Previous studies showed that highly abundant proteins preferentially use cheap (in terms of energy) amino acids. We found that the carbon content is still negatively correlated with protein abundance after controlling for the energetic cost of the amino acids. However, the negative correlation between protein abundance and energetic cost disappeared after controlling for carbon content. Building blocks seem to be more restricted than energy. It seems that the amino acid sequences of highly abundant proteins have to compromise between optimization for their biological functions and reducing the consumption of limiting resources. By contrast, the amino acid sequences of weakly expressed proteins are more likely to be optimized for their biological functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Specific locations of hydrophilic amino acids in constructed transmembrane ligands of the platelet-derived growth factor beta receptor

The 44 amino acid E5 transmembrane protein is the primary oncogene product of bovine papillomavirus. Homodimers of the E5 protein activate the cellular PDGF beta receptor tyrosine kinase by binding to its transmembrane domain and inducing receptor dimerization, resulting in cellular transformation. To investigate the role of transmembrane hydrophilic amino acids in receptor activation, we constructed a library of dimeric small transmembrane proteins in which 16 transmembrane amino acids of the E5 protein were replaced with random, predominantly hydrophobic amino acids. A low level of hydrophilic amino acids was encoded at each of the randomized positions, including position 17, which is an essential glutamine in the wild-type E5 protein. Library proteins that induced transformation in mouse C127 cells stably bound and activated the PDGF beta receptor. Strikingly, 35% of the transforming clones had a hydrophilic amino acid at position 17, highlighting the importance of this position in activation of the PDGF beta receptor. Hydrophilic amino acids in other transforming proteins were found adjacent to position 17 or at position 14 or 21, which are in the E5 homodimer interface. Approximately 22% of the transforming proteins lacked hydrophilic amino acids. The hydrophilic amino acids in the transforming clones appear to be important for driving homodimerization, binding to the PDGF beta receptor, or both. Interestingly, several of the library proteins bound and activated PDGF beta receptor transmembrane mutants that were not activated by the wild-type E5 protein. These experiments identified transmembrane proteins that activate the PDGF beta receptor and revealed the importance of hydrophilic amino acids at specific positions in the transmembrane sequence. Our identification of transformation-competent transmembrane proteins with altered specificity suggests that this approach may allow the creation and identification of transmembrane proteins that modulate the activity of a variety of receptor tyrosine kinases.     

Human red cell galactose 1-phosphate uridylyltransferase: effects of site-specific reagents on catalytic activity

Egg shell membrane protein was found to contain the crosslinking amino acids desmosine and isodesmosine. Of particular interest, the desmosine and isodesmosine content was increased severalfold when the egg shell membrane protein was subjected to autoclaving. The major protein in membranes, which contains the crosslinking amino acids desmosine and isodesmosine, differs greatly from elastin in amino acid composition and is resistant to digestion with elastase. It is concluded that this protein component is not elastin but contains desmosine isomers. Further, its amino acid composition does not resemble those reported for other fibrous proteins such as keratin, connectin, collagen, or microfibrillar protein.     

Resonant recognition model and protein topography. Model studies with myoglobin, hemoglobin and lysozyme

This study describes the further extension of the resonant recognition model for the analysis and prediction of protein--protein and protein--DNA structure/function dependencies. The model is based on the significant correlation between spectra of numerical presentations of the amino acid or nucleotide sequences of proteins and their coded biological activity. According to this physico-mathematical method, it is possible to define amino acids in the sequence which are predicted to be the most critical for protein function. Using sperm whale myoglobin, human hemoglobin and hen egg white lysozyme as model protein examples, sets of predicted amino acids, or so-called 'hot spots', have been identified within the tertiary structure. It was found for each protein that the predicted 'hot spots', which are distributed along the primary sequence, are spatially grouped in a dome-like arrangement over the active site. The identified amino acids did not correspond to the amino acid residues which are involved in the chemical reaction site of these proteins. It is thus proposed that the resonant recognition model helps to identify amino acid residues which are important for the creation of the molecular structure around the catalytic active site and also the associated physical field conditions required for biorecognition, docking of the specific substrate and full biological activity.     

Allosteric Regulation of a Protein Acetyltransferase in Micromonospora aurantiaca by the Amino Acids Cysteine and Arginine

ACT domains (amino acid-binding domains) are linked to a wide range of metabolic enzymes that are regulated by amino acid concentration. Seventy proteins with ACT-GCN5-related N-acetyltransferase (GNAT) domain organization were found in actinomycetales. In this study, we investigate the ACT-containing GNAT acetyltransferase, Micau_1670 (MaKat), from Micromonospora aurantiaca ATCC 27029. Arginine and cysteine were identified as ligands by monitoring the conformational changes that occur upon amino acids binding to the ACT domain in the MaKat protein using FRET assay. It was found that MaKat is an amino acid-regulated protein acetyltransferase, whereas arginine and cysteine stimulated the activity of MaKat with regard to acetylation of acetyl-CoA synthetase (Micau_0428). Our research reveals the biochemical characterization of a protein acetyltransferase that contains a fusion of a GNAT domain with an ACT domain and provides a novel signaling pathway for regulating cellular protein acetylation. These findings indicate that acetylation of proteins and acetyltransferase activity may be tightly linked to cellular concentrations of some amino acids in actinomycetales.     

Comparative characterization of random‐sequence proteins consisting of 5, 12, and 20 kinds of amino acids

Screening of functional proteins from a random‐sequence library has been used to evolve novel proteins in the field of evolutionary protein engineering. However, random‐sequence proteins consisting of the 20 natural amino acids tend to aggregate, and the occurrence rate of functional proteins in a random‐sequence library is low. From the viewpoint of the origin of life, it has been proposed that primordial proteins consisted of a limited set of amino acids that could have been abundantly formed early during chemical evolution. We have previously found that members of a random‐sequence protein library constructed with five primitive amino acids show high solubility (Doi et al., Protein Eng Des Sel 2005;18:279–284). Although such a library is expected to be appropriate for finding functional proteins, the functionality may be limited, because they have no positively charged amino acid. Here, we constructed three libraries of 120‐amino acid, random‐sequence proteins using alphabets of 5, 12, and 20 amino acids by preselection using mRNA display (to eliminate sequences containing stop codons and frameshifts) and characterized and compared the structural properties of random‐sequence proteins arbitrarily chosen from these libraries. We found that random‐sequence proteins constructed with the 12‐member alphabet (including five primitive amino acids and positively charged amino acids) have higher solubility than those constructed with the 20‐member alphabet, though other biophysical properties are very similar in the two libraries. Thus, a library of moderate complexity constructed from 12 amino acids may be a more appropriate resource for functional screening than one constructed from 20 amino acids.     

Metabolic labeling of human primary retinal pigment epithelial cells for accurate comparative proteomics

Metabolic labeling was evaluated, using both 13C6-Arg and 13C6, 15N2-Lys amino acids, for a primary human retinal pigment epithelial cell (hRPE) culture prepared from an autopsy eye of an 81 year old donor. Satisfactory incorporation (>90%) was achieved with both stable isotope labeled amino acids after four passages (roughly 7 population doublings). The degree of incorporation was found to be efficient with both amino acids as well as in different proteins. The presence of 10% whole serum in the culture medium did not interfere with the incorporation of the exogenous stable isotope labeled amino acids. Metabolic labeling of these human primary retinal pigment epithelial cells was further tested to quantify protein ratios between proliferating and resting cells using a combination of 2-DG and MALDI-TOF-TOF/MS analysis. Using computational data processing and analysis, we obtained accurate protein ratio measurement for every single identified protein (156 proteins) in the 2-Dg array. Of these 156 proteins, 12 proteins were found significantly increased in dividing versus resting cells by at least a factor of 1.5 while 13 other proteins were found increased in resting versus dividing cells by at least the same fold. Most of these differentially expressed proteins are directly involved in cell proliferation, protein synthesis, and actin-remodeling and differentiation.     

不同类型氨基酸网络参量与蛋白质折叠的关系

理论和实验研究表明,蛋白质天然拓扑结构对其折叠过程具有重要的影响．采用复杂网络的方法分析蛋白质天然结构的拓扑特征,并探索蛋白质结构特征与折叠速率之间的内在联系．分别构建了蛋白质氨基酸网络、疏水网、亲水网、亲水-疏水网以及相应的长程网络,研究了这些网络的匹配系数(assortativity coefficient)和聚集系数(clustering coefficient)的统计特性．结果表明,除了亲水-疏水网,上述各网络的匹配系数均为正值,并且氨基酸网和疏水网的匹配系数与折叠速率表现出明显的线性正相关,揭示了疏水残基间相互作用的协同性有助于蛋白质的快速折叠．同时,研究发现疏水网的聚集系数与折叠速率有明显的线性负相关关系,这表明疏水残基间三角结构(triangle construction)的形成不利于蛋白质快速折叠．还进一步构建了相应的长程网络,发现序列上间距较远的残基接触对的形成将使蛋白质折叠进程变慢．     

Coding nucleic acids are chaperons for protein folding: A novel theory of protein folding

The arguments for nucleic acid chaperons are reviewed and three new lines of evidence are added. (1) It was found that amino acids encoded by codons in short nucleic acid loops frequently form turns and helices in the corresponding protein structures. (2) The amino acids encoded by partially complementary (1st and 3rd nucleotides) codons are more frequently co-located in the encoded proteins than expected by chance. (3) There are significant correlations between thermodynamic changes (ddG) caused by codon mutations in nucleic acids and the thermodynamic changes caused by the corresponding amino acid mutations in the encoded proteins. We conclude that the concept of the Proteomic Code and nucleic acid chaperons seems correct from the bioinformatics point of view, and we expect to see direct biochemical experiments and evidence in the near future.     

NONRIBOSOMAL INCORPORATION OF AMINO ACIDS INTO THE TROPONIN-LIKE PROTEIN FROM SYNAPTOSOMES

A preparation of synaptosomal cytoplasm was isolated from forebrain of young rats and incubated with various amino acids in vitro. Incorporation of amino acids into protein was observed. This incorporation did not occur by ribosomal protein synthesis. The amino acid incorporating system was not stimulated by ATP and was inhibited by calcium. The system incorporated amino acids enzymatically. An electrophoretic analysis of the synaptosomal preparation, following incubation in the presence of radioactive amino acids, showed only three labelled protein species (molecular weights 37,000, 26,000 and 20,000). This incorporation of amino acids was found to have a high degree of specificity for three protein species. Migration of the three protein species was found to be nearly identical to that of rabbit muscle troponin. The proteins incorporating amino acids were also found to have other characteristics of the troponin subunits. A possible role of troponin modification is discussed.     

The mitochondrial pool of free amino acids reflects the composition of mitochondrial DNA-encoded proteins: indication of a post- translational quality control for protein synthesis

Mitochondria can synthesize a limited number of proteins encoded by mtDNA (mitochondrial DNA) by using their own biosynthetic machinery, whereas most of the proteins in mitochondria are imported from the cytosol. It could be hypothesized that the mitochondrial pool of amino acids follows the frequency of amino acids in mtDNA-encoded proteins or, alternatively, that the profile is the result of the participation of amino acids in pathways other than protein synthesis (e.g. haem biosynthesis and aminotransferase reactions). These hypotheses were tested by evaluating the pool of free amino acids and derivatives in highly-coupled purified liver mitochondria obtained from rats fed on a nutritionally adequate diet for growth. Our results indicated that the pool mainly reflects the amino acid composition of mtDNA-encoded proteins, suggesting that there is a post-translational control of protein synthesis. This conclusion was supported by the following findings: (i) correlation between the concentration of free amino acids in the matrix and the frequency of abundance of amino acids in mtDNA-encoded proteins; (ii) the similar ratios of essential-to-non-essential amino acids in mtDNA-encoded proteins and the mitochondrial pool of amino acids; and (iii), lack of a correlation between codon usage or tRNA levels and amino-acid concentrations. Quantitative information on the mammalian mitochondrial content of amino acids, such as that presented in the present study, along with functional studies, will help us to better understand the pathogenesis of mitochondrial diseases or the biochemical implications in mitochondrial metabolism.     

DNA and amino acid sequence analysis of structural and immunity genes of colicins Ia and Ib.

The nucleotide sequences for colicin Ia and colicin Ib structural and immunity genes were determined. The two colicins each consist of 626 amino acid residues. Comparison of the two sequences along their lengths revealed that the two colicins are nearly identical in the N-terminal 426 amino acid residues. The C-terminal 220 amino acid residues of the colicins are only 60% identical, suggesting that this is the region most likely recognized by their cognate immunity proteins. The predicted proteins for the colicin immunity proteins would contain 111 amino acids for the colicin Ia immunity protein and 115 amino acids for the colicin Ib immunity protein. The colicin immunity proteins have no detectable DNA or amino acid homology but do exhibit a conservation of overall hydrophobicity. The colicin immunity genes lie distal to and in opposite orientation to the colicin structural genes. The colicin Ia immunity protein was purified to apparent homogeneity by a combination of isoelectric focusing and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified Ia immunity protein was determined and was found to be in perfect agreement with that predicted from the DNA sequence of its structural gene. The Ia immunity protein is not a processed membrane protein.     

Kinetics of amino acid incorporation into serum proteins

1. The effect of varying body temperature on the rate of amino acid incorporation into serum protein does not give support to the idea that the rate of this process is adjusted in vivo to restore those protein molecules destroyed by thermal denaturation. The experimentally observed Q₁₀ was about 3.9. 2. When amino acids are injected into the blood of animals in a steady state of serum protein turnover, a period of time elapses before these amino acids can be found in the serum proteins. This has been called transit time. At a given temperature (31°) it is the same in rabbits, turtles, and Limulus (1 hour). In rabbits and turtles it has a Q₁₀ of 3.2. It appears to be specifically related to the process of synthesis (or release) of serum proteins. 3. It was not possible to affect the transit time or the incorporation rate by the administration of amino acid analogues.     

Characteristic sequential residue environment of amino acids in proteins

The occurrence of all di- and tripeptide segments of proteins was counted in a large data base containing about 119 000 residues. It was found that the abundance of the amino acids does not determine the frequency of the various di- and tripeptide segments. In addition, the frequency of the various tripeptides cannot be predicted from the observed pair-frequency values. The pair-frequency distribution of amino acids is highly asymmetrical, pairs formed from identical residues are generally preferred and amino acids cannot be clustered on the basis of their first neighbour preferences. These data indicate the existence of general short range regularities in the primary structure of proteins. The consequences of these short range regularities were studied by comparing Chou-Fasman parameters with analogous parameters determined from the results of conformational energy calculations of single amino acids. This comparison shows that Chou-Fasman parameters carry significant information about the environment of each amino acid. The success of the Chou-Fasman's prediction and the properties of the pair and triplet distribution of the amino acid residues suggest that every amino acid has a characteristic sequential residue environment in proteins. The observed preferences could be invoked, for example, in protein design or in the study of the evolutionary relationship of proteins.     

Kinetic studies on ribosomal proteins assembly in preribosomal particles and ribosomal subunits of mammalian cells.

Proteins were isolated from 80-S preribosomal particles and ribosomal subunits of murine L5178Y cells after short and longer periods of incubation with tritiated amino acids. The labeling patterns of ribosomal proteins were compared by two-dimensional polyacrylamide gel electrophoresis. The analysis of isotopic ratios in individual protein spots showed marked differences in the relative kinetics of protein appearance within nucleolar peribosomes and cytoplasmic subunits. Among the about 60 distinct proteins characterized in 80-S preribosomes, 9 ribosomal proteins appeared to incorporate radioactive amino acids more rapidly. These proteins become labeled gradually in the cytoplasmic ribosomal subunits. It was found that one non-ribosomal protein associated with 80-S preribosomes takes up label far more quickly than other preribosomal polypeptides. It is suggested that this set of proteins could associate early with newly transcribed pre-rRNA, more rapidly than others after their synthesis on polyribosomes, and could therefore play a role in the regulation of ribosome synthesis. In isolated 60-S and 40-S ribosomal subunits, we detected five proteins from the large subunit and four proteins from the small subunit which incorporate tritiated amino acids more quickly than the remainder. These proteins were shown to be absent or very faintly labeled in 80-S preribosomal particles, and would associate with ribosomal particles at later stages of the maturation process.