Long-chain Peptides Research Articles

A Simple Screening and Optimization Bioprocess for Long-Chain Peptide Catalysts Applied to Asymmetric Aldol Reaction.

Peptides have demonstrated their efficacy as catalysts in asymmetric aldol reactions. But the constraints inherent in chemical synthesis have imposed limitations on the viability of long-chain peptide catalysts. A noticeable dearth of tools has impeded the swift and effective screening of peptide catalysts using biological methods. To address this, we introduce a straightforward bioprocess for the screening of peptide catalysts for asymmetric aldol reactions. We synthesized several peptides through this method and obtained a 15-amino acid peptide. This peptide exhibited asymmetric aldol catalytic activity, achieving 77% ee in DMSO solvent and 63% ee with over an 80.8% yield in DMSO mixed with a pH 9.0 buffer solution. The successful application of our innovative approach not only represents an advancement but also paves the way for currently unexplored research avenues.

Structure-Activity Relationship of Novel ACE Inhibitory Undecapeptides from Stropharia rugosoannulata by Molecular Interactions and Activity Analyses.

Undecapeptide is the central peptide molecule in the peptide base material of Stropharia rugosoannulata, and angiotensin-converting enzyme (ACE) plays a crucial role in hypertension. To fully explore the interaction mechanism and ACE-inhibitory activity of long-chain peptides from Stropharia rugosoannulata, the binding conformations of twenty-seven undecapeptides with the ACE receptor were revealed by molecule docking. The undecapeptide GQEDYDRLRPL with better receptor binding capacity and higher secondary mass spectral abundance was screened. All amino acid residues except proline in GQEDYDRLRPL interacted with the ACE receptor. GQEDYDRLRPL interfered with the receptor's overall structure, with significant fluctuations in amino acid residues 340-355, including two residues in the receptor's active pockets. The binding constants of GQEDYDRLRPL to the ACE receptors were at the μM level, with a kinetic binding constant of 9.26 × 10-7 M, which is a strong binding, and a thermodynamic binding constant of 3.06 × 10-6 M. Intermolecular interaction were exothermic, enthalpy-driven, and specific binding reactions. GQEDYDRLRPL had an IC50 value of 164.41 μmol/L in vitro and superior antihypertensive effects at low-gavage administration in vivo. Obtaining information on the interaction mechanism of ACE-inhibitory undecapeptides from S. rugosoannulata with the ACE receptor will help to develop and utilize ACE inhibitors of natural origin.

An improved installation of 2-hydroxy-4-methoxybenzyl (iHmb) method for chemical protein synthesis

The 2-hydroxy-4-methoxybenzyl (Hmb) backbone modification can prevent amide bond-mediated side-reactions (e.g., aspartimide formation, peptide aggregation) by installing the removable Hmb group into a peptide bond, thus improving the synthesis of long and challenging peptides and proteins. However, its use is largely precluded by the limited Hmb's installation sites. In this report, an improved installation of Hmb (iHmb) method was developed to achieve the flexible installation and the convenient removal of Hmb. The iHmb method involves two critical steps: (1) oxidative diazotization of the readily installed 2-hydroxy-4-methoxy-5-amino-benzyl (Hmab) to give 2-hydroxy-4-methoxy-5-diazonium-benzyl (Hmdab) by combining soamyl nitrite (IAN)/HBF4, and (2) reductive elimination of Hmdab to give the desired Hmb by 1,2-ethanedithiol (EDT). The iHmb method enables the installation of Hmb at any primary amino acid including the highly sterically hindered amino acids (e.g., valine and isoleucine). The practicality and utility of the iHmb method was demonstrated by one-shot solid-phase synthesis of a challenging aspartimide-prone peptide, the mirror-image version of a hydrophobic peptide and a long-chain peptide up to 76-residue. Furthermore, the iHmb method can be utilized to facilitate chemical protein ligation, as exemplified by the synthesis of the single-spanning membrane protein sarcolipin. The iHmb method expands the toolkit for peptide synthesis and ligation and facilitates the preparation of peptides/proteins.

Starch digestion retarded by wheat protein hydrolysates with different degrees of hydrolysis

Wheat protein hydrolysates (WPH) were prepared by pepsin hydrolysis for 30, 60, and 120 min (WPH30, WPH60, and WPH120). The mixed system of rice starch and WPH was hydrothermally treated to explore the effect of WPH with different degrees of hydrolysis on starch digestion. WPH reduced the first-order rate coefficient (k) of starch digestion. Especially, WPH30 reduced the k value the most and formed the highest slowly digestible starch content due to the entanglement of starch chains and long-chain peptides. WPH60 and WPH120 with more hydrophobic peptides and polar amino acids than WPH30 tended to form hydrogen bonds with starch molecules due to less steric hindrance. Particularly, the complexation of WPH60 promoted the formation of dense aggregate structure and hindered the enzymatic hydrolysis of starch to a certain extent, thereby increasing the resistant starch content. These findings provide significant guidance for the preparation of hypoglycemic reformed food.

Taste peptides derived from Stropharia rugosoannulata fermentation mycelium and molecular docking to the taste receptor T1R1/T1R3.

This study identified the peptides in the fermentation mycelia of Stropharia rugosoannulata. The molecular weight of the peptides was below 3,000 Da. Heptapeptides to decapeptides were the main peptides in the fermentation mycelia of S. rugosoannulata. More than 50% of the peptides had salty and umami taste characteristics, and the long-chain peptides (decapeptides to 24 peptides) also played an essential role in the pleasant taste characteristics of mycelium. In the salty and umami peptide of S. rugosoannulata, the distribution of non-polar hydrophobic amino acids and polar-uncharged amino acids accounted for a relatively high proportion, and the proportion of polar-uncharged amino acids further increased, with the extension of the peptide chain. P, F, I, l, V, G, S, T, and D were the amino acids with a high proportion in the peptides. The taste peptides can bind to more than 60% of the active amino acid residues in the cavity-binding domain of the T1R1/T1R3 receptors. Hydrogen bond interaction was the primary mode of interaction between the peptides and the receptor. The first and second amino acid residues (such as S, V, E, K, G, and A) at the C-terminal and N-terminal of the peptides were easy to bind to T1R1/T1R3 receptors. Asp108, Asn150, Asp147, Glu301, Asp219, Asp243, Glu70, Asp218 in T1R1, and Glu45, Glu148, Glu301, Glu48, and Ala46 in TIR3 were the key active amino acid sites of taste peptides binding to T1R1/T1R3 receptors.

POTENTIAL OF SCORPION VENOM FOR THE TREATMENT OF VARIOUS DISEASES

The venom of various scorpion species finds significant therapeutic applications. It is rich in neurotoxins, enzymes, enzyme inhibitors, histamine, lipids and different salts from which peptides demonstrate a great potential against a variety of diseases. Many biological functions e. g., bradykinin potentiating, hemolytic, anti-cancer, anti-microbial, and anti-inflammatory potential are being regulated by non-disulfide-bonded peptides. Therefore, it is motivating to use these properties for the treatment of cancer, cardiovascular diseases, diabetes, AIDS, apoplexy, influenza H5N1, paralysis, epilepsy, malaria, measles, severe combined immunodeficiency, fever blisters and diabetes. Scorpion venom has shown the presence of 100,000 bioactive compounds but only 1 % of these have been purified, isolated and characterized by HPLC and mass spectroscopy etc. For the production of high-quality antivenom with specific antibodies, gentler electrical stimulation is a better method as compared to manual production. Recombinant DNA technology has facilitated the identification of new components. Some important medicinal compounds isolated from scorpion venom include HsTX1 (from Heterometrus spinnifer), mucroporin-M1 (from Lychas mucronatus), chlorotoxin and charybdotoxin (from Leiurus quinquestriatus hebraeus). B. leptochelys venom has shown the presence of at least 148 components. Six novel long-chain peptides were isolated from the scorpion Buthus martensi Karsch venom. Crude venom of L. Abdullah bayrami displays a proliferative effect on MCF-7 cells and also shows antimicrobial potential. A new toxin derived from the venom of Liocheles waigiensis [U1-liotoxin-Lw1a (U1-LITX-Lw1a)] displays significant insecticidal action. The computational studies may play an important role while developing ion channel drugs from venom peptides.

Synthesis of Tetrapeptides Containing Dehydroalanine, Dehydrophenylalanine and Oxazole as Building Blocks for Construction of Foldamers and Bioinspired Catalysts.

The incorporation of dehydroamino acid or fragments of oxazole into peptide chain is accompanied by a distorted three-dimensional structure and additionally enables the introduction of non-typical side-chain substituents. Thus, such compounds could be building blocks for obtaining novel foldamers and/or artificial enzymes (artzymes). In this paper, effective synthetic procedures leading to such building blocks—tetrapeptides containing glycyldehydroalanine, glycyldehydrophenylalanine, and glycyloxazole subunits—are described. Peptides containing serine were used as substrates for their conversion into peptides containing dehydroalanine and aminomethyloxazole-4-carboxylic acid while considering possible requirements for the introduction of these fragments into long-chain peptides at the last steps of synthesis.

Protein–water coupling tunes the anharmonicity of amide I modes in the interfacial membrane-bound proteins

The diagonal anharmonicity of an amide I mode of protein backbones plays a critical role in a protein's vibrational dynamics and energy transfer. However, this anharmonicity of long-chain peptides and proteins in H2O environment is still lacking. Here, we investigate the anharmonicity of the amide I band of proteins at the lipid membrane/H2O interface using a surface-sensitive pump-probe setup in which a femtosecond infrared pump is followed by a femtosecond broadband sum frequency generation vibrational spectroscopy probe. It is found that the anharmonicity of the amide I mode in ideal α-helical and β-sheet structures at hydrophobic environments is 3-4cm-1, indicating that the amide I mode in ideal α-helical and β-sheet structures is delocalized over eight peptide bonds. The anharmonicity increases as the bandwidth of the amide I mode increases due to the exposure of peptide bonds to H2O. More H2O exposure amounts lead to a larger anharmonicity. The amide I mode of the peptides with large H2O exposure amounts is localized in one to two peptide bonds. Our finding reveals that the coupling between the amide I mode and the H2O bending mode does not facilitate the delocalization of the amide I mode along the peptide chain, highlighting the impact of H2O on energy transfer and structural dynamics of proteins.

Impact of Intrinsic and Extrinsic Factors on the Pharmacokinetics of Peptides: When Is the Assessment of Certain Factors Warranted?

Peptides are short chains of 2 to 50 amino acids (molecular weight of less than 10 kDa) linked together by peptide bonds. As therapeutic agents, peptides are of interest because the body naturally produces many different peptides. Short-chain peptides have many advantages as compared with long-chain peptides (e.g., low toxicity). The first peptide corticotropin was approved in 1952 for multiple inflammatory diseases and West syndrome. Since then, more than 60 peptides have been approved by the FDA. Pharmacokinetics (PK) is widely used in modern-day drug development for designing a safe and efficacious dose to treat a wide variety of diseases. There are, however, several factors termed as “intrinsic” or “extrinsic” which can influence the PK of a drug, and as a result, one has to adjust the dose in a patient population. These intrinsic and extrinsic factors can be described as age, gender, disease states such as renal and hepatic impairment, drug–drug interaction, food, smoking, and alcohol consumption. It is well known that these intrinsic and extrinsic factors can have a substantial impact on the PK of small molecules, but for macromolecules, the impact of these factors is not well established. This review summarizes the impact of intrinsic and extrinsic factors on the PK of peptides.

Expression and purification of recombinant alpha-toxin AnCra1 from the scorpion Androctonus crassicauda and its functional characterization on mammalian sodium channels.

Alpha-scorpion toxins with long-chain peptide and four disulfide bonds represent diverse pharmacological profiles for various subtypes of voltage-gated sodium channels. Obtaining the natural toxins are difficult and time-consuming process, which represents the major difficulty to interpreting analysis of their structural and functional properties. This study describes the toxin peptide and plasmid construct containing the gene coding for mammalian toxin AnCra1 from the scorpion Androctonus crassicauda venom. We have established genetic construction of fusion protein in pET32a + vector containing thioredoxin (Trx-tag), enterokinase cleavage site and 6xhistidine-tag for efficient expression in Escherichia coli strain RG2 (DE3). The soluble expressed peptide, then purified by Ni-NTA resin affinity chromatography and its purity was confirmed by reverse-phase HPLC and mass spectrometry (7433.54Da.). The electrophysiological data showed that recombinant AnCra1 selectively inhibits the fast inactivation of hNav1.7 channel (EC50 = 136.7 ± 6.6nM). Our findings demonstrate that the AnCra1 is structurally and functionally analogous to alpha excitatory toxins; furthermore, expression and purification of bioactive scorpion toxins in bacterial cells can be a practicable and efficient way to obtain a novel source of toxin peptides as tools to study the function and physiological responses of ion channels.

Supramolecular Antiparallel β-Sheet Formation by Tetrapeptides Based on Amyloid Sequence.

Self-assembly of short peptides has emerged as an interesting research field for a wide range of applications. Recently, several truncated fragments of long-chain peptides or proteins responsible for different neurodegenerative diseases were studied to understand whether they can mimic the property and function of native peptides or not. It was reported that such a kind of peptide adopts a β-sheet structure in the disease state. It was observed that aromatic amino acid-rich peptide fragments possess a high tendency to adopt a β-sheet conformation. In this article, we are first time reporting the crystal structure of two tetrapeptides: Boc-GAII-OMe (Peptide 1) and Boc-GGVV-OMe (Peptide 2), composed of aliphatic amino acids, and the sequences are similar to the Aβ-peptide fragments Aβ29-32 and Aβ37-40 , respectively. In the solid-state, they are self-assembled in an antiparallel β-sheet fashion. The peptide units are connected by the strong amide hydrogen-bonding (N-H···O) interactions. Apart from that, other noncovalent interactions are also present, which help to stabilize the cross-β-sheet arrangement. Interestingly, in the crystal structure of Peptide 1, noncovalent C···C interaction between the electron-deficient carbonyl carbon, and the electron-rich sp3-carbon atom is observed, which is quite rare in the literature. The calculated torsion angles for these peptides are lying in the β-sheet region of the Ramachandran plot. FT-IR studies also indicate the formation of an antiparallel β-sheet structure in the solid-state. Circular dichroism of the peptides in the aqueous solution also suggests the presence of predominantly β-sheet-like conformation in the aqueous solution. Under cross-polarized light, Congo Red stained both peptides showed green-gold color due to birefringence indicating their amyloidogenic nature. This result indicates that the short peptide composed of aliphatic amino acid is capable of forming a β-sheet structure in the absence of aromatic amino acid and also can mimic the function of the native amyloid peptide.

Identification, taste characterization and molecular docking study of novel umami peptides from the Chinese anchovy sauce.

Fish sauce has a subtle flavor with prominent umami and salty taste, and is accompanied by a certain sweetness and bitterness. In order to identify a wider range of umami peptides, Chinese southern and northern anchovy sauce were selected for the study. Seventeen peptides were obtained by separation and purification, and their taste activity was predicted. Through the taste characterization and descriptive analysis, it was found that the synthesized peptides were umami and umami-enhancing peptides. Seventeen umami peptides were simulated and embedded into the umami receptor T1R1/T1R3 by inserting into the Venus flytrap domain (VFTD) of the T1R3 subunit; the interaction forces were mainly hydrogen bonding, electrostatic interaction, van der Waals force and hydrophobic interaction. According to the docking interaction energies, long-chain peptides may be easier to bind to the receptor than short-chain peptides. Asp196, Glu128 and Glu197 were the main binding sites for docking, and could affect umami synergism. For the first time, novel umami peptides in Chinese anchovy sauce have been reported. This study is helpful for discovering umami marine resource peptides, and can provide a basis for further understanding the flavor system of anchovy sauce. © 2020 Society of Chemical Industry.

Characterization and rational design for substrate specificity of a prolyl endopeptidase from Stenotrophomonas maltophilia

A novel prolyl endopeptidase from Stenotrophomonas maltophilia, SmPEP, was discovered and characterized. The specific activity of the recombinant SmPEP expressed by Escherichia coli BL21 (DE3), was 68.3 U/mg at pH 8.0 and 37 °C. In order to improve the substrate specificity for long-chain peptide, rational design was applied based on the structure constructed by homology modeling. Inter-domain sites within the β-propeller domain were chosen for the mutation to weaken the inter-domain interaction and form an open conformation for long-chain substrate entering into the active site. The substrate specificity on a designed long-chain substrate, PQPQLPYPQPQLP, of the mutants F263A and E184 G increased 8.77 and 5.75 times respectively versus wild-type. After the saturated mutation of the both sites, the reactive rate of mutant F263 V on 13-mer peptide was 10.2 times higher than that of the wild-type. Then the mutant F263 V was used in the hydrolysis of casein, and the ACE inhibitory activity of the hydrolysate was significantly improved compared with wild type enzyme, which verified the efficiency of the design strategy.

The variation of two extracellular enzymes and soybean meal bitterness during solid-state fermentation of Bacillus subtilis

The debittering effect of extracellular enzymes from Bacillus subtilis ACCC 01746 was studied using soybean meal as a substrate for solid-state fermentation (SSF). Results showed that B. subtilis produces proteases and carboxypeptidase in the early stage of SSF (0–8 h). Proteases are dominant and can hydrolyze the soybean protein into long-chain peptides with mild bitterness. Carboxypeptidase production is dominant at 8–16 h SSF, at which point soybean protein is further hydrolyzed and bitterness is enhanced. The strain then produces additional carboxypeptidase after 16 h, and bitterness is reduced. We compared the amino acid composition of the hydrolysates from soybean protein isolates to that of the fermented liquid of SSF. In the hydrolysates from soybean protein isolates that exhibit strong bitterness, 62.81% of amino acids are hydrophobic and occur in the form of peptides. In the fermented liquid from soybean meal, 16.22% of amino acids are hydrophobic and are mainly present in the form of free amino acids. The bitterness of fermented soybean hydrolysate is reduced from 5 to 0 when fermented for 24 h, suggesting that B. subtilis can effectively reduce bitterness, possibly due to the carboxypeptidase. Enzyme analysis shows that B. subtilis excretes carboxypeptidase during growth. The amino acids phenylalanine, alanine, tyrosine, and leucine at the C-terminal of the soybean bitter peptides in hydrolysates are cleaved in the presence of carboxypeptidase, resulting in complete debitterness.

Gelatin cages: The formation and characterization of carriers for housing catalyst cargoes

Taking inspiration from nature, research has focused on developing microstructures, nanostructures, and cages constructed from proteins and peptides as appealing devices to compartmentalize and transfer agents and chemical reactions or synthesis. Long-chain peptide and protein cages are scarcely retrieved, and their fabrication is far from clear; nonetheless, they have the advantage of being rigid-chains of high molecular weight, affording diverse supramolecular structures. Here, we report the ability of long gelatin peptides to form monodisperse spheroidal cages. We examine a range of synthetic modality conditions that give rise to gelatin particles with dissimilar properties. Using an emulsion containing a self-organizing system generated by reaction–diffusion, we produce protein cages in a one-vessel process. Based on systematic physiochemical analyses, the upload and release of cargo stored within these cages are convincingly revealed to occur slightly dependently under the conditions of the surrounding environment. We have demonstrated that the gelatin cages entrap silver nanoparticles in their internal volume and in the three-dimensional housing created by the supramolecular structure, allowing to complete the reduction of 4-nitrophenol to 4-aminophenol.

BotAF, a new Buthus occitanus tunetanus scorpion toxin, produces potent analgesia in rodents

This work reports the purification of new potent scorpion neuropeptide, named BotAF, by an activity-guided screening approach. BotAF is a 64-residue long-chain peptide that shares very high similarity with the original β-like scorpion toxin group, in which several peptides have been characterized to be anti-nociceptive in rodents. BotAF administration to rodents does not produce any toxicity or motor impairment, including at high doses. In all models investigated, BotAF turned out to be an efficient peptide in abolishing acute and inflammatory (both somatic and visceral) pain in rodents. It performs with high potency compared to standard analgesics tested in the same conditions. The anti-nociceptive activity of BotAF depends on the route of injection: it is inactive when tested by i.c.v. or i.v. routes but gains in potency when pre-injected locally (in the same compartment than the irritant itself) or by i.t. root 40 to 60 min before pain induction, respectively. BotAF is not an AINS-like compound as it fails to reduce inflammatory edema. Also, it does not activate the opioidergic system as its activity is not affected by naloxone. BotAF does also not bind onto RyR and has low activity towards DRG ion channels (particularly TTX sensitive Na+ channels) and does not bind onto rat brain synaptosome receptors. In somatic and visceral pain models, BotAF dose-dependently inhibited lumbar spinal cord c-fos/c-jun mRNA up regulation. Altogether, our data favor a spinal or peripheral anti-nociceptive mode of action of BotAF.

Biological Portfolio of 1,5-Disubstituted Tetrazoles: A Review

The achievement of medicinal chemistry, in the last few decades, has intensively increased due to the creation of novel drugs withe isostericnature of tetrazole ring with carboxyl group in longer chain peptides was responsible for the medicinal activity. Therefore a comprehensive search was carried out for 1, 5-disubstituted tetrazoles owing to their synthetic and effective pharmacological activities such as anti-inflammatory, antiviral, antibiotic, anti-ulcer, anti-tubercular, anti-hypertensive, etc. In spite of many research papers in the area of tetrazoles with respect to their pharmacological activity there have been no review on1, 5-disubstituted tetrazoles inthis field. The aim of present review is to classify and summarize the pharmaceutical uses of 1, 5-disubstituted tetrazole containing compounds.

Characterization of commercially available peptidases in respect of the production of protein hydrolysates with defined compositions using a three-step methodology

Enzymatic hydrolysis of food proteins is an effective way to implement a desired functionality into protein containing foods. Since these functionalities are highly dependent on the degree of hydrolysis (DH) of the hydrolysates, the enzymatic hydrolysis process must be carefully controlled. Hydrolysates with both free amino acids and long-chain peptides are usually not desirable which leads to strict requirements for the commercial peptidases used. In the study presented, we characterized ten commercially available peptidase preparations in respect of the production of protein hydrolysates with defined compositions. Therefore, we utilized a three-step methodology to investigate the composition of the commercial peptidase preparations and to characterize the obtained protein hydrolysates applying a HPLC-based system for automated small-scale enzymatic reaction and analysis. With the presented methodology we identified two suitable preparations for the production of hydrolysates with high DH and high amounts of free amino acids, and six suitable preparations for the production of hydrolysates with low DH and negligible release of amino acids.

Possibility for Allergy Immunotherapy with Long Synthetic Peptide of Bet V1

In allergy immunotherapy (AIT) using peptides, although the choice of T-cell epitope in consideration of the HLA type is important, application to all HLA types is difficult. We recently devised a new method to address this problem in AIT for white birch pollinosis, using long-chain synthetic peptides by dividing the sequence of Bet v1a, the major allergen in white birch pollen, into three peptides that overlap by 10 amino acid residues. Theoretically, these peptides contain all possible T-cell epitopes for any HLA type. Further, these peptides show weak allergenicity and are considered less prone to cause anaphylaxis because the three-dimensional structures differ significantly from the native Bet v1a. The combination of these three peptides of Bet v1a appears to provide an effective, safe form of AIT for white birch pollinosis. This method may thus be applicable to other allergens.

Proteases supplementation to high gravity worts enhances fermentation performance of brewer's yeast

Nitrogen limitation, particularly prevailing in the case of high gravity beer brewing, results in poor yeast viability and even stuck or sluggish fermentations. Although wort contains abundant proteins and longer chain peptides, brewer's yeast does not assimilate them due to the fact that cells hardly secrete proteases during fermentation. The objective of this study was to investigate the possibility for utilizing unavailable nitrogen from two types of high gravity worts (20°P and 24°P) by adding three food-grade commercial proteases (Neutrase, Flavorzyme and Protamex) at the beginning of fermentations, respectively. Results showed that proteases supplementation significantly increased the FAN level and thus the amount of cell suspension in the later stages of fermentations (ca. 10days later for 20°P and 25 days later for 24°P) (p<0.05). Among the studied three proteases, we found that fermentations with Flavorzyme supplementation exhibited the best fermentation performance in terms of significantly improved wort fermentability, higher ethanol yield and flavor volatiles formation (p<0.05). Furthermore, the foam of final beers produced by adding proteases was as stable as that of the control at each of the corresponding gravities.