Construction of protocellular structures under simulated primitive earth conditions

We have developed experimental approaches for the construction of protocellular structures under simulated primitive earth conditions and studied their formation and characteristics. Three types of envelopes; protein envelopes, lipid envelopes, and lipid-protein envelopes are considered as candidates for protocellular structures. Simple protein envelopes and lipid envelopes are presumed to have originated at an early stage of chemical evolution, interaction mutually and then evolved into more complex envelopes composed of both lipids and proteins.Three kinds of protein envelopes were constructedin situ from amino acids under simulated primitive earth conditions such as a fresh water tide pool, a warm sea, and a submarine hydrothermal vent. One protein envelope was formed from a mixture of amino acid amides at 80 °C using multiple hydration-dehydration cycles. Marigranules, protein envelope structures, were produced from mixtures of glycine and acidic, basic and aromatic amino acids at 105 °C in a modified sea medium enriched with essential transition elements. Thermostable microspheres were also formed from a mixture of glycine, alanine, valine, and aspartic acid at 250 °C and above. The microspheres did not form at lower temperatures and consist of silicates and peptide-like polymers containing imide bonds and amino acid residues enriched in valine. Amphiphilic proteins with molecular weights of 2000 were necessary for the formation of the protein envelopes.Stable lipid envelopes were formed from different dialkyl phospholipids and fatty acids.Large, stable, lipid-protein envelopes were formed from egg lecithin and the solubilized marigranules. Polycations such as polylysine and polyhistidine, or basic proteins such as lysozyme and cytochromec also stabilized lipid-protein envelopes.     

Comparative Study of the Conformational Lock,Dissociative Thermal Inactivation and Stability of <Emphasis Type="Italic">Euphorbia</Emphasis> Latex and Lentil Seedling Amine Oxidases

The thermal stability of copper/quinone containing amine oxidases from Euphorbia characias latex (ELAO) and lentil seedlings (LSAO) was measured in 100 mM potassium phosphate buffer (pH 7.0) following changes in absorbance at 292 nm. ELAO was shown to be about 10°C more stable than LSAO. The dissociative thermal inactivation of ELAO was studied using putrescine as substrate at different temperatures in the range 47–70°C, and a “conformational lock” was developed using the theory pertaining to oligomeric enzyme. Moreover ELAO was shown to be more stable towards denaturants than LSAO, as confirmed by dodecyl trimethylammonium bromide denaturation curves. A comparison of the numbers of contact sites in inter-subunits of ELAO relative to LSAO led us to conclude that the higher stability of ELAO to temperature and towards denaturants was due to the presence of larger number of contact sites in the conformational lock of the enzyme. This study also gives a putative common mechanism for thermal inactivation of amine oxidases and explains the importance of C-terminal conserved amino acids residues in this class of enzymes.     

Early life relict feature in peptide mass distribution

The molecular mass of a biomolecule is characterized by the monoisitopic mass M_mono and the average isotopic mass M_av. We found that tryptic peptide masses mapped on a plane made by two parameters derived from M_mono and M_av form a peculiar feature in the form of a ‘band gap’ stretching across the whole ‘peptide galaxy’, with a narrow line in the centre. The purpose of this study was to investigate possible reasons for the emergence of such a feature, provided it is not a random occurrence. The a priori probability of such a feature to emerge by chance was found to be less than 1:100. Peptides contributing to the central line have elemental compositions following the rules S = 0; Z = C - (N + H)/2 = 0, which nine out of 20 amino acid residues satisfy. The relative abundances of amino acids in the peptides contributing to the central line correlate with the consensus order of emergence of these amino acids, with ancient amino acids being overrepresented in on-Line peptides. Since linear correlation between M_av and M_mono reduces the complexity of polypeptide molecules, and the turnover rate of less complex molecules should be faster in non-equilibrium abiotic synthesis, we hypothesize that the line could be a signature of abiotic production of primordial biopolymers. The linear dependence between the average isotopic masses and monoisotopic masses may have influenced the selection of amino acid residues for terrestrial life.     

Control of stable RNA synthesis in a temperature-sensitive mutant of elongation factor G of Bacillus subtilis

Summary A temperature-sensitive EFG mutant of Bacillus subtilis was isolated and characterized. This mutant, ts32, synthesizes stable RNA at 48° C with or at 50° C without accompanied protein synthesis. The initial rate of the RNA synthesis at 48° C or 50° C was 1.5 to 2.0 times as much as that at 30° C.This mutant as well as its parent (both leu ^-) showed stringent response for the RNA synthesis upon deprivation of amino acids with an accumulation of the MS nucleotides (pp Gpp and pppGpp). On raising temperature to 48° C or 50° C, the ts-cells immediately began to synthesize the stable RNA with an initial increase of the MS nucleotides. No drastic decrease in amount of the MS was observed during the active RNA synthesis.These results suggest that EFG is somehow involved in repressing the stable RNA synthesis, and have broken the close relationship between the stable RNA synthesis and the MS nucleotides hitherto reported.     

Peptide Amyloids in the Origin of Life

How life can emerge from non-living matter is one of the fundamental mysteries of the universe. A bottom-up approach to this problem focuses on the potential chemical precursors of life, in particular the nature of the first replicative molecules. Such thinking has led to the currently most popular idea: that an RNA-like molecule played a central role as the first replicative and catalytic molecule. Here, we review an alternative hypothesis that has recently gained experimental support, focusing on the role of amyloidogenic peptides rather than nucleic acids, in what has been by some termed “the amyloid-world” hypothesis. Amyloids are well-ordered peptide aggregates that have a fibrillar morphology due to their underlying structure of a one-dimensional crystal-like array of peptides in a β-strand conformation. While they are notorious for their implication in several neurodegenerative diseases including Alzheimer's disease, amyloids also have many biological functions. In this review, we will elaborate on the following properties of amyloids in relation to their fitness as a prebiotic entity: they can be formed by very short peptides with simple amino acids sequences; as aggregates they are more chemically stable than their isolated component peptides; they can possess diverse catalytic activities; they can form spontaneously during the prebiotic condensation of amino acids; they can act as templates in their own chemical replication; they have a structurally repetitive nature that enables them to interact with other structurally repetitive biopolymers like RNA/DNA and polysaccharides, as well as with structurally repetitive surfaces like amphiphilic membranes and minerals.     

Synthesis of stable C‐linked ferrocenyl amino acids and their use in solution‐phase peptide synthesis

Incorporation of ferrocenyl group to peptides is an efficient method to alter their hydrophobicity. Ferrocenyl group can also act as an electrochemical probe when incorporated onto functional peptides. Most often, ferrocene is incorporated onto peptides post‐synthesis via amide, ester or triazole linkages. Stable amino acids containing ferrocene as a C‐linked side chain are potentially useful building units for the synthesis of ferrocene‐containing peptides. We report here an efficient route to synthesize ferrocene‐containing amino acids that are stable and can be used in peptide synthesis. Coupling of 2‐ferrocenyl‐1,3‐dithiane and iodides derived from aspartic acid or glutamic acid using n‐butyllithium leads to the incorporation of a ferrocenyl unit to the δ‐position or ε‐position of an α‐amino acid. The reduction or hydrolysis of the dithiane group yields an alkyl or an oxo derivative. The usability of the synthesized amino acids is demonstrated by incorporating one of the amino acids in both C‐terminus and N‐terminus of tripeptides in solution phase. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Evidence in the formation of conjugated linoleic acids from thermally induced 9t12t linoleic acid: a study by gas chromatography and infrared spectroscopy

Thermally induced isomerisation leading to the formation of conjugated linoleic acids (CLAs) has been observed for the first time during the thermal treatment of 9t12t fatty acid triacylglycerol, and methyl ester. Fifteen microlitre portions of the triacylglycerol sample containing 9t12t fatty acid (trilinoelaidin) were placed in micro glass ampoules and sealed under nitrogen, then subjected to thermal treatment at 250 °C. The glass ampoules were removed at regular time intervals, cut open, and the contents were analysed by infrared spectroscopy using a single reflectance attenuated total internal reflectance crystal accessory. The samples were then subjected to derivatisation into their methyl esters. The methyl esters of the isomerised fatty acids were analysed by gas chromatography. The same procedure was repeated with methyl ester samples containing 9t12t fatty acid (methyl linoelaidate). Each sample was subjected to infrared measurements and gas chromatographic analysis after appropriate dilution in heptane.The results show that the thermally induced isomerisation of 9t12t fatty acids from both triacylglycerol molecules and methyl esters give identical CLA profiles as those found for the thermally induced isomerisation of 9c12c fatty acids. The infrared spectrometry provides additional evidence confirming the formation of CLA acids during thermal treatment. A mechanism for the formation of the CLAs from 9t12t fatty acid molecules is also formulated for the first time. This mechanism complements the pathways of formation of CLAs from 9c12c fatty acids during thermal treatment.     

Characterization of the N-linked high-mannose oligosaccharides of the insulin pro-receptor and mature insulin receptor subunits.

An interesting pattern in the genetic code was reported previously [Blalock & Smith (1984) Biochem. Biophys. Res. Commun. 121, 203-207]. In the 5'-to-3' direction, codons for hydrophilic and hydrophobic amino acids are generally complemented by codons for hydrophobic and hydrophilic amino acids respectively. The average tendency of codons for 'unchanged' (slightly hydrophilic) amino acids was to be complemented by codons for 'unchanged' amino acids. We now show that the same pattern results when the complementary codon is read in the 3'-to-5' direction. This pattern is further shown to result in the interaction of peptides specified by complementary RNAs regardless of whether the amino acids are assigned in the 5'-to-3' or the 3'-to-5' direction. Here we demonstrate that peptides specified by complementary RNAs bind to each other with specificity and high affinity.     

Polyarginine enters cells more efficiently than other polycationic homopolymers.

Homopolymers or peptides containing a high percentage of cationic amino acids have been shown to have a unique ability to cross the plasma membrane of cells, and consequently have been used to facilitate the uptake of a variety of biopolymers and small molecules. To investigate whether the polycationic character of these molecules, or some other structural feature, was the molecular basis for the effect, the ability of a variety of homopolymers to enter cells was assayed by confocal microscopy and flow cytometry. Polymers of L- or D-arginine containing six or more amino acids entered cells far more effectively than polymers of equal length composed of lysine, ornithine and histidine. Peptides of fewer than six amino acids were ineffective. The length of the arginine side-chain could be varied without significant loss of activity. These data combined with the inability of polymers of citrulline to enter cells demonstrated that the guanidine headgroup of arginine was the critical structural component responsible for the biological activity. Cellular uptake could be inhibited by preincubation of the cells with sodium azide, but not by low temperature (3 degrees C), indicating that the process was energy dependent, but did not involve endocytosis.     

Cutting edge: HLA-B27 acquires many N-terminal dibasic peptides: coupling cytosolic peptide stability to antigen presentation

Ag presentation by MHC class I is a highly inefficient process because cytosolic peptidases destroy most peptides after proteasomal generation. Various mechanisms shape the MHC class I peptidome. We define a new one: intracellular peptide stability. Peptides with two N-terminal basic amino acids are more stable than other peptides. Such peptides should be overrepresented in the peptidome of MHC class I-associated peptides. HLA-B27 binding peptides use anchor residue R at P2 and, although most amino acids are allowed, particular amino acids are overrepresented at P1, including R and K. We show that such N-terminal dibasic peptides are indeed more efficiently presented by HLA-B27. This suggests that HLA-B27 can present peptides from Ags present in fewer copies than required for successful peptide generation for other MHC class I molecules.     

Phylogeny-based design of a B-subunit of DNA gyrase and its ATPase domain using a small set of homologous amino acid sequences

We have developed a phylogeny-based design method that has been used to produce mutated proteins with enhanced thermal stabilities. We previously validated the predictive worth of the method by producing and characterizing mutants in which one original residue or a small number of the original residues had been replaced with the one or the ones found in the phylogenetically predicted “ancestral” sequence. For the current study, this method was used to design a sequence for the deepest nodal position of a phylogenic tree composed of 16 gyrase B-subunit sequences, which was then synthesized and characterized. The sequence was inferred from the sequences of 16 extant DNA gyrases and 3 extant type VI DNA topoisomerases. Genes encoding the inferred sequence and its N-terminal ATPase domain were PCR constructed and expressed in Escherichia coli. The full-length designed protein is slightly less thermally stable than is subunit B from the extant thermophilic Thermus thermophilus DNA gyrase, whereas the thermal stability of the designed ATPase domain is more similar to that of the T. thermophilus ATPase domain. Moreover, the designed ATPase domain has significant catalytic activity. Therefore, even a small set of homologous amino acid sequences contains sufficient information to design a thermally stable and functional protein. Because the isolated designed ATPase domain is more thermally stable and catalytically active than is the sequence containing the most frequently occurring amino acids among the 16 gyrases, the phylogenetic approach was superior (in this case, at least) to the consensus approach when the same data set was used to predict the two sequences.     

Solid phase isotope exchange with spillover hydrogen in amino acids,peptides, and proteins

We summarize here information on the theoretical and experimental study of high-temperature (150–200°C) solid phase catalytic isotope exchange (HSCIE) carried out with amino acids, peptides, and proteins under the action of spillover hydrogen. Main specific features of the HSCIE reaction, its mechanism, and its use for studying spatial interactions in polypeptides are discussed. A virtually complete absence of racemization makes this reaction a valuable preparative method. The main regularities of the HSCIE reaction with the participation of spillover tritium have been revealed in the case of peptides and proteins, and the dependence of reactivity of peptide fragments on the spatial organization of their molecules has been studied. An important peculiarity of this reaction is that HSCIE proceeds at 150–200°C with a high degree of chirality retention in amino acids and peptides. This is provided by its reaction mechanism, which consists in a synchronous one-center substitution at the saturated carbon atom characterized by the formation of pentacoordinated carbon and a three-center bond between the carbon and the incoming and outgoing hydrogen atoms.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 3–21.Original Russian Text Copyright © 2005 by Zolotarev, Dadayan, Borisov.     

Chemotactic behavior of Campylobacter spp. in function of different temperatures (37 °C and 42 °C)

The chemotactic behaviour of Campylobacter strains was determined in the presence of different amino acids at two temperatures (37 °C and 42 °C). Two strains of catalase positive (Campylobacter jejuni) and negative (Campylobacter sputurum) Campylobacter were isolated from river water in Tonekabon, Iran and identified by phenotyping and 16srRNA Gene sequencing methods. Chemotactic responses of the isolates were assessed toward a variety of amino acids viz., L-cystine, L-asparagine, L-histidine, L-aspartic acid, L-serine, L-phenylalanine, L-leucine and L-tryptophan by disc and capillary methods at two temperatures: 37 °C and 42 °C. C. jejuni showed positive chemotactic response towards L-cystine,L-tryptophan, L-phenylalanine, - L-leucine, L-asparagine and L-Serine at both, 37 °C and 42 °C however, it was greater at 37 °C. C. sputurum showed negative or weak response towards all of the amino acids. In addition, C. jejuni illustrated strong chemotactic response to L-asparagine follow by L-serine and weak chemotaxis response to L-phenylalanine and L-cysteine at 37 °C. Overall, C. jejuni showed relatively strong chemotactic response to some amino acids, likewise it was greater at 37 °C. Hence, the human body temperature (37 °C) in compared to avian body temperature (42 °C) probably promotes chemotactic response of C. jejuni, which it might be a reason for causing disease in human being compared to avian.     

Origin of life: Consideration of alternatives to proteins and nucleic acids

Summary Starting with relatively simple, non-hydrolyzable compounds in aqueous solution, entirely spontaneous condensations give rise to polymers that contain purines, pyrimidines, amino acids, coenzymes, lipid components and even phosphate. The presence of certain lipid micelles allows significant product formation at millimolar substrate concentrations. The first step involves formation of a Michael adduct from--unsaturated carbonyl compounds and various nucleophiles. Polymerization of these adducts occurs via sequential Knoevenagel condensations. All reactions take place readily at temperatures below 45°. The polymers can act as macromolecular catalysts as evidenced by hydrolytic activity. The purines and pyrimidines in the polymers appear to be capable of both base pairing and stacking interactions with ribonucleic acids. Specific examples of potential alternatives to base pairing are presented. These results are discussed from the standpoint of the spontaneous development of reproducing molecules. Proteins and nucleic acids may be evolutionary developments which have displaced earlier biopolymers.     

Effect of constant and fluctuating temperatures on resting and active oxygen consumption of toads,Bufo boreas

Summary The relations of standard and active rates of oxygen consumption to body temperature (T_b) were tested in montane Bufo b. boreas and lowland Bufo boreas halophilus acclimated to constant T _b of 10, 20, or 30° C or to a fluctuating cycle of 5–30° C. Standard metabolic rates (SMR) of boreas acclimated to 30° C and halophilus acclimated to 10° C show pronounced regions of thermal independence but all other standard and active metabolic rates of groups acclimated to other thermal regimes are thermally sensitive. The SMR of both subspecies acclimated to the 5–30° C cycle are more thermally sensitive than those of similar individuals acclimated to constant T _b. In cases where the relation between SMR and T _b is linear for both halophilus and boreas at the same acclimation temperature, the slope and Q₁₀ of the relation for boreas are significantly higher than those of halophilus. Acclimation had little or no effect on the active metabolic rates of either subspecies. The relation between SMR and T _b of boreas maintained under field conditions (Carey, 1979) is matched only by those of individuals from the same population acclimated to 20° C.     

Phenylalanine-Binding RNAs and Genetic Code Evolution

We isolated RNAs by selection–amplification, selecting for affinity to Phe–Sepharose and elution with free l-phenylalanine. Constant sequences did not contain Phe condons or anticodons, to avoid any possible confounding influence on initially randomized sequences. We examined the eight most frequent Phe-binding RNAs for inclusion of coding triplets. Binding sites were defined by nucleotide conservation, protection, and interference data. Together these RNAs comprise 70% of the 105 sequenced RNAs. The K _D for the strongest sites is ≈50 μM free amino acid, with strong stereoselectivity. One site strongly distinguishes free Phe from Trp and Tyr, a specificity not observed previously. In these eight Phe-binding RNAs, Phe codons are not significantly associated with Phe binding sites. However, among 21 characterized RNAs binding Phe, Tyr, Arg, and Ile, containing 1342 total nucleotides, codons are 2.7-fold more frequent within binding sites than in surrounding sequences in the same molecules. If triplets were not specifically related to binding sites, the probability of this distribution would be 4.8 × 10⁻¹¹. Therefore, triplet concentration within amino acid binding sites taken together is highly likely. In binding sites for Arg, Tyr, and Ile cognate codons are overrepresented. Thus Arg, Tyr, and Ile may be amino acids whose codons were assigned during an era of direct RNA–amino acid affinity. In contrast, Phe codons arguably were assigned by another criterion, perhaps during later code evolution.     

Construction of chiral polyesters from polycondensation of multifunctional monomer containing both flexible amino acid and rigid pendant groups with aromatic diols

A number of chiral wholly aromatic polyesters (PEs) with phthalimido and flexible chiral unit in the backbone were prepared from a chiral synthesized diacid monomer, 5-(3-methyl-2-phthalimidylpentanoylamino)isophthalic acid (1), and various aromatic diols via the polyesterification reaction. The tosyl chloride/pyridine/N,N-dimethylformamide (DMF) system was used as a condensing agent. All of the these polymers having bulky phthalimido and amino acid functionalities in the side chain showed excellent solubility and readily dissolved in various solvents such as N-methyl-2-pyrrolidinone, N,N-dimethylacetamide and DMF. Since, these chiral polymers have natural amino acids in the polymer architecture, they are expected to be biodegradable and therefore may be classified under eco-friendly polymers. They had useful levels of thermal stability associated with excellent solubility. Thermogravimetric analysis (TGA) showed that the obtained PEs are rather thermally stable, 10% weight loss temperatures in excess of 317°C, and char yields at 700°C in the nitrogen atmosphere higher than 24%. The resulting polymers were obtained in good yields with inherent viscosities ranging between 0.22 and 0.56 dL/g and were characterized with FT-IR, ¹H-NMR, elemental and TGA techniques.     

Thermal preference and activity thresholds in populations of Argia vivida (Odonata: Coenagrionidae) from habitats with different thermal regimes

The hypothesis was tested that isolated populations of Argia vivida Hagen living in habitats with different thermal regimes would show similar larval temperature preferenda, similar distributions in a temperature gradient, similar larval upper temperature acitivity thresholds, and similar adult minimum temperature flight thresholds. The hypothesis was supported in all cases, except for distribution within the gradient, where there were significantly fewer observations below 22°C in a population from a habitat with a fluctuating diel and annual temperature regime than in a population from a more thermally stable habitat. Larval modal temperature preferendum was 28°C; escape temperature (EST) was 35.4–36.4°C, critical thermal maximum (CTM) was 39.1–41.0°C, and upper lethal temperature (ULT) was 44.4–46.0°C. While technical difficulties affected the estimates of flight thresholds, there was no difference between the field estimates from different sites. Minimum body temperature for flight appears to be about 25°C, apparently higher than for several other zygopterans, while larval activity thresholds and 96 hr LD50 of 36.8°C are similar to those recorded for other odonates.     

Robustness of predictions of extremely thermally stable proteins in ancient organisms

A number of studies have addressed the environmental temperatures experienced by ancient life. Computational studies using a nonhomogeneous evolution model have estimated ancestral G + C contents of ribosomal RNAs and the amino acid compositions of ancestral proteins, generating hypotheses regarding the mesophilic last universal common ancestor. In contrast, our previous study computationally reconstructed ancestral amino acid sequences of nucleoside diphosphate kinases using a homogeneous model and then empirically resurrected the ancestral proteins. The thermal stabilities of these ancestral proteins were equivalent to or greater than those of extant homologous thermophilic proteins, supporting the thermophilic universal ancestor theory. In this study, we reinferred ancestral sequences using a dataset from which hyperthermophilic sequences were excluded. We also reinferred ancestral sequences using a nonhomogeneous evolution model. The newly reconstructed ancestral proteins are still thermally stable, further supporting the hypothesis that the ancient organisms contained thermally stable proteins and therefore that they were thermophilic.     

Photosynthetic 14CO2 fixation products in altitudinal ecotypes of Trifolium repens L. with different temperature requirements

Summary Two Trifolium repens clones from natural meadows at 600 m and 2030 m above sea level, and with differing dependence on temperature of their rate of apparent photosynthesis, were grown under controlled environments. Radioactive products in detached leaves were examined after 20 and 40 s periods of steady state photosynthesis in ¹⁴CO₂ at 3° C and 24° C. Glycine and serine were hardly labeled at 3° C. At 24° C, the leaves of the alpine clone showed significantly, (P<0.025) more activity in these amino acids than those from the low altitude clone. It is suggested that the alpine clone has a higher photorespiration. This is supported by the labeling pattern of glucose, fructose, sucrose, and glucose-6-phosphate.The high altitude clone requires lower temperatures for photosynthesis than the low altitude clone. It is suggested, that this is caused by its higher photorespiration, which reduces net photosynthesis at high temperatures. The lower photorespiration activity of the low altitude clone can be interpreted as an adaptation to its warmer habitat.