Are prions a relic of an early stage of peptide evolution?

The rather unique properties of prions and their presence in very different kinds of living species suggest that this type of molecule was created at a very early stage of evolution and may even represent a relic from a time where peptide evolution was ongoing and RNA/DNA did not yet exist. A comparison of the most frequently occurring amino acid sequences in known prions with the sequences preferentially formed in the salt-induced peptide formation reaction, the most simple mechanism enabling the formation of peptides under primitive earth conditions, shows a remarkable coincidence that strongly supports this hypothesis.     

Are prions a relic of an early stage of peptide evolution?

The rather unique properties of prions and their presence in very different kinds of living species suggest that this type of molecule was created at a very early stage of evolution and may even represent a relic from a time where peptide evolution was ongoing and RNA/DNA did not yet exist. A comparison of the most frequently occurring amino acid sequences in known prions with the sequences preferentially formed in the salt-induced peptide formation reaction, the most simple mechanism enabling the formation of peptides under primitive earth conditions, shows a remarkable coincidence that strongly supports this hypothesis.     

Methionine peptide formation under primordial earth conditions

According to recent research on the origin of life it seems more and more likely that amino acids and peptides were among the first biomolecules formed on earth and that a peptide/protein world was thus a key starting point in evolution towards life. Salt-induced Peptide Formation (SIPF) has repeatedly been shown to be the most universal and plausible peptide-forming reaction currently known under prebiotic conditions and forms peptides from amino acids with the help of copper ions and sodium chloride. In this paper we present experimental results for salt-induced peptide formation from methionine. This is the first time that a sulphur-containing amino acid was investigated in this reaction. The possible catalytic effects of glycine and l-histidine in this reaction were also investigated and a possible distinction between the l- and d-forms of methionine was studied as well.     

Attachment of peptide building blocks to proteins through tyrosine bioconjugation

Recent efforts have yielded a number of short peptide sequences with useful binding, sensing, and cellular uptake properties. In order to attach these sequences to tyrosine residues on intact proteins, a three-component Mannich-type strategy is reported. Two solid phase synthetic routes were developed to access peptides up to 20 residues in length with anilines at either the N- or C-termini. In the presence of 20 mM formaldehyde, these functional groups were coupled to tyrosine residues on proteins under mild reaction conditions. The identities of the resulting bioconjugates were confirmed using mass spectrometry and immunoblot analysis. Screening experiments have demonstrated that the method is compatible with substrates containing all of the amino acids, including lysine and cysteine residues. Importantly, tyrosine residues on proteins exhibit much faster reaction rates, allowing short peptides containing this residue to be coupled without cross reactions.     

Investigations on the mechanism of the salt-induced peptide formation

The applicability of the salt-induced peptide formation in aqueous solution — the simplest model so far for peptide synthesis under primitive earth conditions — is demonstrated for valine as another amino acid, and the formation of mixed peptides in systems containing glycine, alanine and valine is investigated. The dominant dipeptides formed are Gly-Gly, Gly-Ala and Gly-Val, at longer reaction times sequence inversion produces Ala-Gly and, considerably slower, Val-Gly. Ala-Ala is also produced and the relative amounts of the diastereomers prove the high conservation of optical purity of the original amino acids over a considerable time. The results lead to some further conclusions about the reaction mechanism and the possible dominance of peptide sequences in primordial dipeptides.     

Catalytic effects of glycine on prebiotic divaline and diproline formation

The catalytic effects of the simple amino acid glycine on the formation of diproline and divaline in the prebiotically relevant salt-induced peptide formation (SIPF) reaction was investigated in systems of different amino acid starting concentrations and using the two enantiomeric forms of the respective amino acid. Results show an improved applicability of the SIPF reaction to prebiotic conditions, especially at low amino acid concentrations, as presumably present in a primordial scenario, and indicate excellent conditions and resources for chemical evolution of peptides and proteins on the early earth. For valine, furthermore differences in catalytic yield increase are found indicating a chiral selectivity of the active copper complex of the reaction and showing a connection to previously found enantiomeric differences in complex formation constants with amino acids.     

Preferential amino acid sequences in alumina-catalyzed peptide bond formation

The catalytic effect of activated alumina on amino acid condensation was investigated. The readiness of amino acids to form peptide sequences was estimated on the basis of the yield of dipeptides and was found to decrease in the order glycine (Gly), alanine (Ala), leucine (Leu), valine (Val), proline (Pro). For example, approximately 15% Gly was converted to the dipeptide (Gly(2)), 5% to cyclic anhydride (cyc(Gly(2))) and small amounts of tri- (Gly(3)) and tetrapeptide (Gly(4)) were formed after 28 days. On the other hand, only trace amounts of Pro(2) were formed from proline under the same conditions. Preferential formation of certain sequences was observed in the mixed reaction systems containing two amino acids. For example, almost ten times more Gly-Val than Val-Gly was formed in the Gly+Val reaction system. The preferred sequences can be explained on the basis of an inductive effect that side groups have on the nucleophilicity and electrophilicity, respectively, of the amino and carboxyl groups. A comparison with published data of amino acid reactions in other reaction systems revealed that the main trends of preferential sequence formation were the same as those described for the salt-induced peptide formation (SIPF) reaction. The results of this work and other previously published papers show that alumina and related mineral surfaces might have played a crucial role in the prebiotic formation of the first peptides on the primitive earth.     

Conformational analysis of invariant peptide sequences in bacterial genomes

The functional significance of evolutionarily conserved motifs/patterns of short regions in proteins is well documented. Although a large number of sequences are conserved, only a small fraction of these are invariant across several organisms. Here, we have examined the structural features of the functionally important peptide sequences, which have been found invariant across diverse bacterial genera. Ramachandran angles (phi,psi) have been used to analyze the conformation, folding patterns and geometrical location (buried/exposed) of these invariant peptides in different crystal structures harboring these sequences. The analysis indicates that the peptides preferred a single conformation in different protein structures, with the exception of only a few longer peptides that exhibited some conformational variability. In addition, it is noticed that the variability of conformation occurs mainly due to flipping of peptide units about the virtual C(alpha)...C(alpha) bond. However, for a given invariant peptide, the folding patterns are found to be similar in almost all the cases. Over and above, such peptides are found to be buried in the protein core. Thus, we can safely conclude that these invariant peptides are structurally important for the proteins, since they acquire unique structures across different proteins and can act as structural determinants (SD) of the proteins. The location of these SD peptides on the protein chain indicated that most of them are clustered towards the N-terminal and middle region of the protein with the C-terminal region exhibiting low preference. Another feature that emerges out of this study is that some of these SD peptides can also play the roles of "fold boundaries" or "hinge nucleus" in the protein structure. The study indicates that these SD peptides may act as chain-reversal signatures, guiding the proteins to adopt appropriate folds. In some cases the invariant signature peptides may also act as folding nuclei (FN) of the proteins.     

Using peptide arrays to define nuclear carrier binding sites on nucleoporins

In the peptide SPOT array technique, an array of different peptides are synthesized on, and covalently linked to, cellulose membranes. In one usage of this technique, these peptides are screened in an overlay assay to determine which short sequence(s) contains a binding site for an interacting protein. By preparing overlapping peptides that cover the entire sequence of a protein, all of the binding domains on the protein for a second protein can be identified. We have utilized the peptide SPOT array technique to identify the short amino acid sequences within nuclear pore complex proteins (also known as nucleoporins or Nups) that bind the nuclear carrier importin-beta. Crystallization studies by others have indicated that nuclear carriers such as importin-beta bind to phenylalanine-glycine (FG) repeats present in numerous copies in the sequences of a family of nucleoporins. Consistent with this, we found that most (but not all) of the Nup binding sites for importin-beta identified by this technique contain Fx, FG, FxFG, FxFx, or GLFG sequences, although not all such sequences bound importin-beta. Peptide SPOT array substitution studies confirmed a crucial role for the phenylalanine in FG repeats and identified a lysine residue flanking some repeats that is crucial for importin-beta binding to those repeats. In addition to these expected binding sequences for importin-beta, we found multiple instances of a peptide lacking a canonical FG repeat that strongly bound importin-beta, indicating that additional Nup sequences may form binding sites for importin-beta.     

运用 mRNA 体外展示技术筛选胸苷酸合成酶 RNA 亲和肽

以体外选择方法筛选不同功能的核酸、肽和蛋白质是近年的研究热点, mRNA 体外展示是一种新兴的高效多肽选择技术,其基本原理是通过含嘌呤霉素寡核苷酸的 Linker 使 mRNA 与它编码的肽或蛋白质共价结合,形成 mRNA- 蛋白质融合体,这一方法已用于多种功能肽的鉴定 . 以 mRNA 体外展示技术进行了由大容量多肽库中 (>1013) 筛选胸苷酸合成酶 (thymidylate synthase , TS) RNA 亲和肽的研究,通过精密的实验设计,建立了一套完整有效的筛选方法,并对实验条件进行了优化 . 已进行了 8 轮筛选,结果表明,以 mRNA 体外展示技术获得的多肽分子,可以与 TS mRNA 亲和 . 将测序结果与初始肽库进行比较,发现亲和肽中碱性氨基酸及芳香族氨基酸含量明显增加,说明其在与 RNA 结合中具有重要作用 . mRNA 展示技术作为一种大容量文库的体外筛选方法,将广泛应用于与固定化靶物质具高度亲和性及特异性的多肽和蛋白质的筛选 .     

Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phage-displayed peptide library

Microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTG) has been used in many industrial applications because it effectively catalyzes the formation of covalent cross-linking between glutamine residues in various substrate proteins and lysine residues or primary amines. To better understand the sequence preference around the reactive glutamine residue by this enzymatic reaction, we screened preferred peptide sequences using a phage-displayed random peptide library. Most of the peptides identified contained a consensus sequence, which was different from those previously found for mammalian TGases. Of these, most sequences had a specific reactivity toward MTG when produced as a fusion protein with glutathione-S-transferase. Furthermore, the representative sequence was found to be reactive even in the peptide form. The amino acid residues in the sequence critical for the reactivity were further analyzed, and the possible interaction with the enzyme has been discussed in this paper.     

Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins

One mechanism for the spontaneous degradation of polypeptides is the intramolecular attack of the peptide bond nitrogen on the side chain carbonyl carbon atom of aspartic acid and asparagine residues. This reaction results in the formation of succinimide derivatives and has been shown to be largely responsible for the racemization, isomerization, and deamidation of these residues in several peptides under physiological conditions (Geiger, T. & Clarke, S. J. Biol. Chem. 262, 785-794 (1987]. To determine if similar reactions might occur in proteins, I examined the sequence and conformation about aspartic acid and asparagine residues in a sample of stable, well-characterized proteins. There did not appear to be any large bias against dipeptide sequences that readily form succinimides in small peptides. However, it was found that aspartyl and asparaginyl residues generally exist in native proteins in conformations where the peptide bond nitrogen atom cannot approach the side chain carbonyl carbon to form a succinimide ring. These orientations also represent energy minimum states, and it appears that this factor may account for a low rate of spontaneous damage to proteins by succinimide-linked reactions. The presence of aspartic acid and asparagine residues in other conformations, such as those in partially denatured, conformationally flexible regions, may lead to more rapid succinimide formation and contribute to the degradation of the molecule. The possible role of isoimide intermediates, formed by the attack of the peptide oxygen atom on the side chain carboxyl group, in protein racemization, isomerization, and deamidation is also considered.     

Identification of peptides mimicking the ligands of proteins phosphorylated by protein kinase CK2

Synthetic peptides containing a phosphorylation site for protein kinase CK2 were used to investigate their binding properties to other peptides/proteins. The aim of this work was to find an efficient procedure to search for these peptide/protein ligands. The goal was successfully achieved through screening of random peptide libraries displayed on phage. Peptides corresponding to the amino terminal region of topoisomerase I were synthesized in both phosphorylated and unphosphorylated form and used to screen the libraries. Four of the selected sequences were also tested for their reactivity with synthetic peptides corresponding to the carboxy terminal region of the largest subunit of RNA polymerase II. The positive reaction detected supports the hypothesis that the isolated sequences may represent mimics of ligands of proteins phosphorylated by protein kinase CK2.     

Amyloid fibril formation in vitro from halophilic metal binding protein: Its high solubility and reversibility minimized formation of amorphous protein aggregations

Halophilic proteins are characterized by high net negative charges and relatively small fraction of hydrophobic amino acids, rendering them aggregation resistant. These properties are also shared by histidine‐rich metal binding protein (HP) from moderate halophile, Chromohalobacter salexigens, used in this study. Here, we examined how halophilic proteins form amyloid fibrils in vitro. His‐tagged HP, incubated at pH 2.0 and 58°C, readily formed amyloid fibrils, as observed by thioflavin fluorescence, CD spectra, and transmission or atomic force microscopies. Under these low‐pH harsh conditions, however, His‐HP was promptly hydrolyzed to smaller peptides most likely responsible for rapid formation of amyloid fibril. Three major acid‐hydrolyzed peptides were isolated from fibrils and turned out to readily form fibrils. The synthetic peptides predicted to form fibrils in these peptide sequences by Waltz software also formed fibrils. Amyloid fibril was also readily formed from full‐length His‐HP when incubated with 10–20% 2,2,2‐trifluoroethanol at pH 7.8 and 25°C without peptide bond cleavage.     

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.     

Peptide Formation Mechanism on Montmorillonite Under Thermal Conditions

The oligomerization of amino acids is an essential process in the chemical evolution of proteins, which are precursors to life on Earth. Although some researchers have observed peptide formation on clay mineral surfaces, the mechanism of peptide bond formation on the clay mineral surface has not been clarified. In this study, the thermal behavior of glycine (Gly) adsorbed on montmorillonite was observed during heating experiments conducted at 150 °C for 336 h under dry, wet, and dry–wet conditions to clarify the mechanism. Approximately 13.9 % of the Gly monomers became peptides on montmorillonite under dry conditions, with diketopiperazine (cyclic dimer) being the main product. On the other hand, peptides were not synthesized in the absence of montmorillonite. Results of IR analysis showed that the Gly monomer was mainly adsorbed via hydrogen bonding between the positively charged amino groups and negatively charged surface sites (i.e., Lewis base sites) on the montmorillonite surface, indicating that the Lewis base site acts as a catalyst for peptide formation. In contrast, peptides were not detected on montmorillonite heated under wet conditions, since excess water shifted the equilibrium towards hydrolysis of the peptides. The presence of water is likely to control thermodynamic peptide production, and clay minerals, especially those with electrophilic defect sites, seem to act as a kinetic catalyst for the peptide formation reaction.     

Mutual Amino Acid Catalysis in Salt-Induced Peptide Formation Supports this Mechanism's Role in Prebiotic Peptide Evolution

The presence of some amino acids and dipeptides under the conditions of the salt-induced peptide formation reaction (aqueous solution at 85 °C, Cu(II) and NaCl) has been found to catalyze the formation of homopeptides of other amino acids, which are otherwise produced only in traces or not at all by this reaction. The condensation of Val, Leu and Lys to form their homodipeptides can occur to a considerable extent due to catalytic effects of other amino acids and related compounds, among which glycine, histidine, diglycine and diketopiperazine exhibit the most remarkable activity. These findings also lead to a modification of the table of amino acid sequences preferentially formed by the salt-induced peptide formation (SIPF) reaction, previously used for a comparison with the sequence preferences in membrane proteins of primitive 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.     

On the chemical basis of the Lowry protein determination

The copper-catalyzed oxidation of peptides and proteins by phosphomolybdic/phosphotungstic acid (Folin phenol reagent) was studied with respect to redox stoichiometry of color formation and nature of the oxidation products. From peptides without reducing side chains two reducing equivalents were transferred under ideal conditions to Mo6+/W6+ for each unit of tetradentate copper complex with concomitant formation of an imino peptide. Tyrosine and tryptophan side chains contributed four additional reducing equivalents. Oxidation of proline-containing peptides was greatly impaired as judged from color formation due to the interference of the imino acid with complex formation. Reaction of the oxidized peptides with 2,4-dinitrophenyl (DNP)-hydrazine gave a peptide amine and the DNP-hydrazone of a 2-oxoacyl peptide. The oxidation products from tetraalanine were identified as dialanine amide and pyruvoylalanine DNP-hydrazone. From the time course of the development of the blue color on reduction of Folin reagent with tetraalanine it was inferred that the reaction consisted of an initial (less than 5 s) oxidation to a Cu3+ peptide complex followed by slow changes in absorbance, especially above 0.2 mM. Due to these complications the two-electron stoichiometry has to be considered only as a limiting case for peptide concentrations below 0.02 mM.