Dynamic Co-evolution of Peptides and Chemical Energetics, a Gateway to the Emergence of Homochirality and the Catalytic Activity of Peptides

We propose a scenario for the dynamic co-evolution of peptides and energy on the primitive Earth. From a multi component system consisting of hydrogen cyanide, several carbonyl compounds, ammonia, alkyl amine, carbonic anhydride, borate and isocyanic acid, we show that the reversibility of this system leads to several intermediate nitriles, that irreversibly evolve to alpha-amino acids and N-carbamoyl amino acids via selective catalytic processes. On the primitive Earth these N-carbamoyl amino acids combined with energetic molecules (NOx) may have been the core of a molecular engine producing peptides permanently and assuring their recycling and evolution. We present this molecular engine, a production example, and its various selectivities. The perspectives for such a dynamic approach to the emergence of peptides are evoked in the conclusion.     

Delivery of extraterrestrial amino acids to the primitive Earth. Exposure experiments in Earth orbit.

A large collection of micrometeorites has been recently extracted from Antarctic old blue ice. In the 50 to 100 micrometers size range, the carbonaceous micrometeorites represent 80% of the samples and contain 2% of carbon. They might have brought more carbon to the surface of the primitive Earth than that involved in the present surficial biomass. Amino acids such as "-amino isobutyric acid have been identified in these Antarctic micrometeorites. Enantiomeric excesses of L-amino acids have been detected in the Murchison meteorite. A large fraction of homochiral amino acids might have been delivered to the primitive Earth via meteorites and micrometeorites. Space technology in Earth orbit offers a unique opportunity to study the behaviour of amino acids required for the development of primitive life when they are exposed to space conditions, either free or associated with tiny mineral grains mimicking the micrometeorites. Our objectives are to demonstrate that porous mineral material protects amino acids in space from photolysis and racemization (the conversion of L-amino acids into a mixture of L- and D-molecules) and to test whether photosensitive amino acids derivatives can polymerize in mineral grains under space conditions. The results obtained in BIOPAN-1 and BIOPAN-2 exposure experiments on board unmanned satellite FOTON are presented.     

D-Amino Acids in Living Higher Organisms

The homochirality of biological amino acids (L-amino acids) andof the RNA/DNA backbone (D-ribose) might have become establishedbefore the origin of life. It has been considered that D-aminoacids and L-sugars were eliminated on the primitive Earth.Therefore, the presence and function of D-amino acids in livingorganisms have not been studied except for D-amino acids in thecell walls of microorganisms. However, D-amino acids wererecently found in various living higher organisms in the form offree amino acids, peptides, and proteins. Free D-aspartate andD-serine are present and may have important physiologicalfunctions in mammals. D-amino acids in peptides are well knownas opioid peptides and neuropeptides. In protein, D-aspartateresidues increase during aging. This review deals with recentadvances in the study of D-amino acids in higher organisms.     

The catalytic effect of L- and D-histidine on alanine and lysine peptide formation

A starting phase of chemical evolution on our ancient Earth around 4 billion years ago was the formation of amino acids and their combination to peptides and proteins. The salt-induced peptide formation (SIPF) reaction has been shown to be appropriate for this condensation reaction under moderate and plausible primitive Earth conditions, forming short peptides from amino acids in aqueous solution containing sodium chloride and Cu(II) ions. In this paper we report results about the formation of dialanine and dilysine from their monomers in this reaction. The catalytic influence of l- and d-histidine dramatically increases dialanine yields when starting from lower alanine concentrations, but also dilysine formation is markedly boosted by these catalysts. Attention is paid to measurable preferences for one enantiomeric form of alanine and lysine in the SIPF reaction. Alanine, especially, shows stereospecific behaviour, mostly in favour of the l-form.     

Cyanamide mediated syntheses under plausible primitive earth conditions

The synthesis of palmitoylglycerols in good yields occurs when a solution of glycerol, ammonium palmitate, cyanamide and imidazole is dried and heated at ambient humidity at temperatures ranging from 60 degrees--100 degrees C for 16 h. Much less product is formed in the absence of either or both cyanamide or imidazole. This work suggests that acylglycerols could have been synthesized on the primitive Earth under plausible prebiotic conditions which were similar but not identical to those which have been shown to condense deoxynucleotides into oligodeoxynucleotides and amino acids into peptides.     

Cloning,expression, and assembly of sericin-like protein

Recombinant sericin proteins of different molecular masses (17.4, 31.9, and 46.5 kDa), based on the 38-amino acid repetitive motif of native sericin, were cloned, expressed, and purified. The recombinant sericin self-assembled during dialysis (starting concentration of 2.5 mg/ml) forming twisted fibers. Circular dichroism and Fourier transform infrared spectroscopy studies demonstrated protein conformational transitions occurred from random coil to beta-sheets during the dialysis. Congo red-stained recombinant sericin fibrils exhibited apple-green birefringence, indicating long-range order in the array of beta-sheets. Biosynthetic sericin has a high content of polar amino acids (e.g. > 40 mol % serine), leading to a beta-sheet conformation formed by hydrogen bonding via polar zipper interactions. Analysis of recombinant sericin sequence using Mandel-Gutfreund's (Mandel-Gutfreund, Y., and Gregoret, L. M. (2002) J. Mol. Biol. 323, 453-461) definition of polar and non-polar amino acids showed that the hydrophobicity pattern resembles the most frequent pattern of amyloidogenic proteins, polar amino acid aggregates (PPPPP). Many beta-proteins and peptides are designed to study amyloidogenesis using a polar/non-polar alternating pattern (PNPNPN). Sericin-like proteins or peptides provide an alternative model in terms of hydrophobicity pattern with which to explore questions related to beta-sheet formation and amyloidogenesis. The glue-like property of sericin is attributed to the hydrogen bonding between serine residues of sericin with serine residues in the fibroin structural components of silk fiber.     

The design of linear peptides that fold as monomeric beta-sheet structures.

Current knowledge about the determinants of beta-sheet formation has been notably improved by the structural and kinetic analysis of model peptides, by mutagenesis experiments in proteins and by the statistical analysis of the protein structure database (Protein Data Bank; PDB). In the past year, several peptides comprising natural and non-natural amino acids have been designed to fold as monomeric three-stranded beta-sheets. In all these cases, the design strategy has involved both the statistical analysis of the protein structure database and empirical information obtained in model beta-hairpin systems and in proteins. Only in one case was rotamer analysis performed to check for the compatibility of the sidechain packing. It is foreseeable that, in future designs, algorithms exploring the sequence and conformational space will be employed. For the design of small proteins (less than 30 amino acids), questions remain about the demonstration of two-state behavior, the formation of a well-defined network of mainchain hydrogen bonds and the quantification of the structured populations.     

Artificial insertion of peptides between signal peptide and mature protein: effect on secretion and processing of hybrid thermostable alpha-amylases in Bacillus subtilis and Escherichia coli cells

To study the effect of inserted peptides on the secretion and processing of exported proteins in Bacillus subtilis and Escherichia coli, pBR322-derived DNA fragments coding for small peptides were inserted between the DNA coding for the 31 amino acid B. subtilis alpha-amylase signal peptide and that coding for the mature part of the extracellular thermostable alpha-amylase of B. stearothermophilus. Most of the inserted peptides (21 to 65 amino acids) decreased the production of the enzyme in B. subtilis and E. coli, the effect of each peptide being similar in the two strains. In contrast, with one peptide (a 21 amino acid sequence encoded by the extra DNA in pTUBE638), the production of alpha-amylase was enhanced more than 1.7-fold in B. subtilis in comparison with that of the parent strain. The molecular masses of the thermostable alpha-amylases in the periplasm of the E. coli transformants varied for each peptide insert, whereas those in the culture supernatants of the B. subtilis transformants had molecular masses similar to that of the mature enzyme. Based on the NH2-terminal amino acid sequence of the hybrid protein from pTUBE638, it was shown that in E. coli, the NH2-terminally extended thermostable alpha-amylase was translocated and remained in the periplasm after the 31 amino acid signal sequence was removed. In the case of B. subtilis, after the removal of a 34-amino acid signal sequence, the hybrid protein was secreted and processed to the mature form.     

Beta-sheet capping: signals that initiate and terminate beta-sheet formation

In the present work, we address the question of whether different amino acids have different beta-sheet initiating and terminating characteristics. Using a large scale analysis of parallel and antiparallel beta-sheets in a non-redundant dataset of proteins, we observed that most of the amino acids show significant under- or over-representation in at least one of the positions at the two ends of beta-sheets, which are denoted as N-cap and C-cap. In addition, based on statistical data and structural comparison, we found that certain amino acids, especially Asp, Asn, Gly and Pro have strong tendencies to block beta-sheet continuation. Hence, we can consider these residues as beta-sheet terminators. It was also proposed that the dipole moments in parallel beta-sheets, whose direction is from C-terminal (partially negative) to N-terminal (partially positive), are much stronger than has previously been suggested. In fact, enhancement of dipole moments in parallel beta-sheets is a result of the positioning of positively charged residues at N-cap and negatively charged residues at C-cap. This enhancement in dipole moment magnitude leads to strengthened dipolar interactions between parallel beta-sheets dipoles and other partners especially alpha-helices dipoles. The results provide an explanation for the antiparallel alignment of parallel beta-sheets with alpha-helices.     

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.     

Discovering structural correlations in alpha-helices.

We have developed a new representation for structural and functional motifs in protein sequences based on correlations between pairs of amino acids and applied it to alpha-helical and beta-sheet sequences. Existing probabilistic methods for representing and analyzing protein sequences have traditionally assumed conditional independence of evidence. In other words, amino acids are assumed to have no effect on each other. However, analyses of protein structures have repeatedly demonstrated the importance of interactions between amino acids in conferring both structure and function. Using Bayesian networks, we are able to model the relationships between amino acids at distinct positions in a protein sequence in addition to the amino acid distributions at each position. We have also developed an automated program for discovering sequence correlations using standard statistical tests and validation techniques. In this paper, we test this program on sequences from secondary structure motifs, namely alpha-helices and beta-sheets. In each case, the correlations our program discovers correspond well with known physical and chemical interactions between amino acids in structures. Furthermore, we show that, using different chemical alphabets for the amino acids, we discover structural relationships based on the same chemical principle used in constructing the alphabet. This new representation of 3-dimensional features in protein motifs, such as those arising from structural or functional constraints on the sequence, can be used to improve sequence analysis tools including pattern analysis and database search.     

Dissolved Divalent Metal and pH Effects on Amino Acid Polymerization: A Thermodynamic Evaluation

Polymerization of amino acids is a fundamentally important step for the chemical evolution of life. Nevertheless, its response to changing environmental conditions has not yet been well understood because of the lack of reliable quantitative information. For thermodynamics, detailed prediction over diverse combinations of temperature and pH has been made only for a few amino acid–peptide systems. This study used recently reported thermodynamic dataset for the polymerization of the simplest amino acid “glycine (Gly)” to its short peptides (di-glycine and tri-glycine) to examine chemical and structural characteristics of amino acids and peptides that control the temperature and pH dependence of polymerization. Results showed that the dependency is strongly controlled by the intramolecular distance between the amino and carboxyl groups in an amino acid structure, although the side-chain group role is minor. The polymerization behavior of Gly reported earlier in the literature is therefore expected to be a typical feature for those of α-amino acids. Equilibrium calculations were conducted to examine effects of dissolved metals as a function of pH on the monomer–polymer equilibria of Gly. Results showed that metals shift the equilibria toward the monomer side, particularly at neutral and alkaline pH. Metals that form weak interaction with Gly (e.g., Mg²⁺) have no noticeable influence on the polymerization, although strong interaction engenders significant decrease of the equilibrium concentrations of Gly peptides. Considering chemical and structural characteristics of Gly and Gly peptides that control their interactions with metals, it can be expected that similar responses to the addition of metals are applicable in the polymerization of neutral α-amino acids. Neutral and alkaline aqueous environments with dissolved metals having high affinity with neutral α-amino acids (e.g., Cu²⁺) are therefore not beneficial places for peptide bond formation on the primitive Earth.     

The origin of the biologically coded amino acids

Biology uses essentially 20 amino acids for its coded protein enzymes, representing a very small subset of the structurally possible set. Most models of the origin of life suggest organisms developed from environmentally available organic compounds. A variety of amino acids are easily produced under conditions which were believed to have existed on the primitive Earth or in the early solar nebula. The types of amino acids produced depend on the conditions which prevailed at the time of synthesis, which remain controversial. The selection of the biological set is likely due to chemical and early biological evolution acting on the environmentally available compounds based on their chemical properties. Once life arose, selection would have proceeded based on the functional utility of amino acids coupled with their accessibility by primitive metabolism and their compatibility with other biochemical processes. Some possible mechanisms by which the modern set of 20 amino acids was selected starting from prebiotic chemistry are discussed.     

Prebiotic phosphate: a problem insoluble in water ? ]

It is well-known that in water phosphate readily reacts with calcium, precipitating as insoluble apatite. How phosphorus could have been available for prebiotic reactions is still an open problem. We suggest that phosphorus-containing compounds might have accumulated in a hydrophobic medium, since the absence of calcium ions would have prevented them from precipitating as apatite. Hydrophobic compounds may have been synthesized on the early Earth through the polymerization of methane or through Fischer-Tropsch-type reactions. Moreover, hydrophobic compounds would have been delivered to the early Earth by extraterrestrial infall. In previous articles (Morchio and Traverso [1999], Morchio et al. [2001]) we suggested that such hydrophobic material would have formed a hydrophobic layer on the surface of the sea, which would have provided an environment thermodynamically more suitable than water for the concentration and polymerization of organic molecules fundamental to life, particularly amino acids and (pyrimidine) bases. It may be hypothesized that elemental phosphorus or phosphorus-containing compounds (such as phosphite) deriving from volcanic eruptions would have ended up raining down into the hydrophobic layer, accumulating due to the absence of calcium ions, in an environment protected against hydrolysis. Phosphorus-containing compounds might have interacted with hydrophobic molecules in the layer giving rise to polymers. In particular, phosphite might have reacted with the hydrophobic amino acids, giving rise to phosphoamino acids, which, in turn, might have interacted with pyrimidine bases (relatively abundant in the layer) giving rise to peptides and oligonucleotide-like polymers. Indeed, it has been experimentally shown (Zhou et al. [1996]) that, in an anhydrous organic medium (pyridine), dialkilphosphite reacts with amino acids to form phosphoamino acids, which interact with pyrimidine nucleosides to give nucleotides, short oligonucleotides and phosphoryl peptides.     

Polymerization of amino acids under primitive earth conditions

Summary Chemical evolution on the primitive earth must have involved the condensation of-amino acids to peptides under a variety of conditions. Subjecting a mixture of methane, ammonia, and water to an electric discharge in the presence of free amino acids yields small peptides. The dehydration-condensation may have taken place via ammonium cyanide, the hydrogen cyanide tetramer, or aminonitriles. The experiments may be considered genuinely prebiotic and significant in the context of chemical evolution.     

Role of hydrophobic clusters in the stability of alpha-helical coiled coils and their conversion to amyloid-like beta-sheets

We designed a library of short peptides using standard rules for coiled-coil assembly. Depending on the composition of amino acids in the non-interacting region of the coiled coil (positions b, c, and f) these peptides are able to convert from alpha-helical to beta-sheet secondary structure. This type of transition is observed in amyloid-like proteins and is a key feature associated with many types of neurodegenerative diseases. Studies on peptides that are 14 amino acids in length indicated that positioning hydrophobic amino acids at an f position within a heptad repeat accelerated the rate of conformational conversion as compared to that at a c position. We believe that this occurs because of the formation of a hydrophobic pocket that preferentially stabilizes beta-sheets over alpha-helices. This effect was also observed in longer 21 amino acid peptides. Our study shows that the relative rates of structural conversion correlate with the formation of a continuous three-amino-acid hydrophobic patch consisting of amino acids in the d, f, and a positions and not on the secondary structure propensities of the individual amino acids. The sequence-structure relationship observed in this study will be used to help understand the mechanism of amyloid fiber formation and design future coiled-coil and beta-sheet-forming peptide systems.     

Folding of beta-sheets in membranes: specificity and promiscuity in peptide model systems

The interactions that drive the folding of beta-barrel membrane proteins have not been well studied because there have been few available model systems for membrane beta-sheets. In this work, we expand on a recently described model system to explore the contributions of interstrand hydrogen bonds, side-chain/side-chain interactions and side-chain/membrane interactions to beta-sheet formation in membranes. These experiments are based on the observation that the hydrophobic hexapeptide acetyl-Trp-Leu-Leu-Leu-Leu-Leu-OH (AcWLLLLL) folds, cooperatively and reversibly, into oligomeric, antiparallel beta-sheets in phosphatidylcholine membranes. To systematically characterize the important interactions that drive beta-sheet formation in membranes, we have used circular dichroism spectroscopy to determine the membrane secondary structure of each member of a complete host-guest family of related peptides of the form AcWLL-X-LL, where X is one of the natural amino acids. Peptides with hydrophobic X-residues of any size or character (X=Ala, Val, Ile, Leu, Cys, Met, Phe and Trp) form similar beta-sheets in membranes, while peptides with any polar X-residue or Gly or Pro at the X-position are random-coils, even when bound to membranes at high concentrations. The observed membrane sheet preferences correlate poorly with intrinsic sheet propensity scales measured in soluble proteins, but they correlate well with several membrane hydrophobicity scales. These results support the idea that the predominant interactions of the side-chains in membrane-bound beta-sheets are with the membrane lipids, and that backbone hydrogen bonding is the major driving force for the stabilization of beta-sheets in membranes.     

A procedure to verify an amino acid sequence which has been derived from a nucleotide sequence: application to the 26S RNA of Semliki Forest virus.

We describe a peptide sequencing procedure which can be used to verify an amino acid sequence which is derived from a nucleotide sequence. One first labels the protein with a 3H- and a 14C-labelled amino acid and then cleaves the protein into a set of peptides using a cleavage reaction specific for a particular amino acid residue. Finally one performs Edman degradations on the whole mixture of peptides. The released amino acids reflect the combined aminoterminal amino acid sequences of all the peptides that have been formed by the cleavage reaction. The data can therefore be used to check a deduced sequence simultaneously at several regions of the polypeptide chain. We have applied this sequencing procedure to verify the amino acid sequence deduced from the 26S RNA of Semliki Forest virus.     

Polynucleotide replication coupled to protein synthesis: A possible mechanism for the origin of life

A mechanism is suggested for the replication under primitive conditions of long polynucleotides by the sequential incorporation of sequences related to those of modern transfer RNAs. It is proposed that replication of such molecules became established as the result of a replicative advantage arising from the concomitant linkage together of amino acids to form polypeptides. Initially these polypeptides may have been of random sequence. Selection of primitive tRNAs in which the amino acid and anticodon stem sequences were rotaionally symmetrical could have led to specific, anticodon-directed aminoacylation and fixation of the genetic code along the lines suggested by Hopfield. (Hopfield, 1978). The primitive replication-coupled system would then have been able to synthesize specific proteins containing one amino acid residue for each primitive tRNA incorporated during replication. The end result of this line of evolution is postulated to have been a nucleoprotein structure resembling the ribosome. The primitive system would then have been able to give rise directly to triplet-coded protein synthesis. Some recent RNA sequence data are discussed which are consistent with derivation of modern protein synthesis from the primitive replication-coupled mechanism.     

Protein sequence analysis studies on the low molecular weight hydrophobic proteins associated with bovine pulmonary surfactant

Lipid extracts of bovine pulmonary surfactant, which exhibit biophysical and biological activity, contain two hydrophobic proteins which have been designated surfactant protein-B (SP-B) and SP-C. Amino terminal amino acid sequence analysis of whole lipid extracts and partially purified protein fractions gave rise to three sequences, two major and one minor. The first sequence, identified as a member of the SP-B family, extended for 60 amino acids beginning with an amino terminal phe. The second polypeptide, identified as a member of the SP-C family, sequenced for 35 amino acids and had a leu amino terminus. The third minor sequence corresponded to amino acids 2-9 of SP-C (N-leu) and was designated SP-C (N-ile). Sequence analysis of cyanogen bromide peptides derived from methyl isocyanate-blocked lipid extract material produced two peptides which extended the amino acid sequence of SP-B to residue 79, which appears to be a glycine.