Peptide Bond Formation Research Articles

Use of Magnetic Carbon Nanocomposites in the Formation of a Recognition Layer of a Piezoelectric Immunosensor for the Determination of Penicillin G

Conditions for the formation of a recognition layer of a piezoelectric immunosensor based on magnetic carbon nanocomposites (MCNCs) under the action of an external magnetic field are studied. The effects of the size and number of magnetic nanoparticles (MNPs) in the composite on the analytical characteristics of the gravimetric immunosensor are revealed. Scanning electron microscopy is used to determine the average sizes of Fe3O4 magnetic nanoparticles synthesized by coprecipitation. It is noted that the minimum weight and stability of the recognition layer were observed for the nanocomposite obtained at a ratio of carbon nanotubes and MNPs with an average diameter of 22 nm equal to 3 : 1. The formation of peptide bonds between the MCNCs and a penicillin G conjugate was established by IR spectrometry. It was shown that the use of magnetic carbon nanocomposites in the formation of a recognition layer makes it possible to significantly simplify the procedure for preparing a piezoelectric sensor for analysis and reduce its duration from 24 to 1.5 h. The range of the determined antibiotic concentrations is 1–450 ng/mL, the limit of detection is 0.5 ng/mL.

Peptide Bond Formation in the Protonated Serine Dimer Following Vacuum UV Photon‐Induced Excitation

AbstractPossible routes for intra‐cluster bond formation (ICBF) in protonated serine dimers have been studied. We found no evidence of ICBF following low energy collision‐induced dissociation (in correspondence with previous works), however, we do observe clear evidence for ICBF following photon absorption in the 4.6–14 eV range. Moreover, the comparison of photon‐induced dissociation measurements of the protonated serine dimer to those of a protonated serine dipeptide provides evidence that ICBF, in this case, involves peptide bond formation (PBF). The experimental results are supported by ab initio molecular dynamics and exploration of several excited state potential energy surfaces, unraveling a pathway for PBF following photon absorption. The combination of experiments and theory provides insight into the PBF mechanisms in clusters of amino acids, and reveals the importance of electronic excited states reached upon UV/VUV light excitation.

Peptide Bond Formation in the Protonated Serine Dimer Following Vacuum UV Photon-Induced Excitation.

Possible routes for intra-cluster bond formation (ICBF) in protonated serine dimers have been studied. We found no evidence of ICBF following low energy collision-induced dissociation (in correspondence with previous works), however, we do observe clear evidence for ICBF following photon absorption in the 4.6-14 eV range. Moreover, the comparison of photon-induced dissociation measurements of the protonated serine dimer to those of a protonated serine dipeptide provides evidence that ICBF, in this case, involves peptide bond formation (PBF). The experimental results are supported by ab initio molecular dynamics and exploration of several excited state potential energy surfaces, unraveling a pathway for PBF following photon absorption. The combination of experiments and theory provides insight into the PBF mechanisms in clusters of amino acids, and reveals the importance of electronic excited states reached upon UV/VUV light excitation.

An Unusual Acceleration of Amination Reactivity by the Proximal Ester Groups in Catechol Diesters: An Efficient Way for Peptide Synthesis

Peptide bond formation has drawn more and more attention due to its importance in medicinal chemistry and drug discovery. Using active esters to construct peptide bonds is an indispensable strategy in this field. We herein report a fast and efficient amination reaction to synthesize peptides from catechol diesters. Catechol monoesters are good active esters in peptide synthesis due to the anchimeric assistance effect. In previously reported work, the amination reaction was inactivated if the 2-hydroxyl group in the catechol esters was functionalized. However, we found an unusual acceleration of the amination reactivity when the 2-hydroxyl group was functionalized as an ester group. The reaction can be complete in 2–3 min in some cases. This acceleration was found through experimental studies to not result from the anchimeric assistance effect. We proposed a π*−π* interaction pathway between the two proximal ester groups. Some computational studies were made to support this assumption.

Consensus design and engineering of an efficient and high-yield peptide asparaginyl ligase for protein cyclization and ligation

Plant legumains are Asn/Asp-specific endopeptidases (AEPs) that have diverse functions in plants. Peptide asparaginyl ligases (PALs) are a special legumain subtype that primarily catalyze peptide bond formation rather than hydrolysis. PALs are versatile protein engineering tools but are rarely found in nature. To overcome this limitation, here we describe a two-step method to design and engineer a high-yield and efficient recombinant PAL based on commonly found AEPs. We first constructed a consensus sequence derived from 1,500 plant legumains to design the evolutionarily stable legumain conLEG that could be produced in E. coli with 20-fold higher yield relative to that for natural legumains. We then applied the LAD (ligase-activity determinant) hypothesis to exploit conserved residues in PAL substrate-binding pockets and convert conLEG into conPAL1-3. Functional studies showed that conLEG is primarily a hydrolase, whereas conPALs are ligases. Importantly, conPAL3 is a super-efficient and broadly active PAL for protein cyclization and ligation.

Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome.

The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3'-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

Elongation Factor P Is Important for Sporulation Initiation.

The universally conserved protein elongation factor P (EF-P) facilitates translation at amino acids that form peptide bonds with low efficiency, particularly polyproline tracts. Despite its wide conservation, it is not essential in most bacteria and its physiological role remains unclear. Here, we show that EF-P affects the process of sporulation initiation in the bacterium Bacillus subtilis. We observe that the lack of EF-P delays expression of sporulation-specific genes. Using ribosome profiling, we observe that expression of spo0A, encoding a transcription factor that functions as the master regulator of sporulation, is lower in a Δefp strain than the wild type. Ectopic expression of Spo0A rescues the sporulation initiation phenotype, indicating that reduced spo0A expression explains the sporulation defect in Δefp cells. Since Spo0A is the earliest sporulation transcription factor, these data suggest that sporulation initiation can be delayed when protein synthesis is impaired. IMPORTANCE Elongation factor P (EF-P) is a universally conserved translation factor that prevents ribosome stalling at amino acids that form peptide bonds with low efficiency, particularly polyproline tracts. Phenotypes associated with EF-P deletion are pleiotropic, and the mechanistic basis underlying many of these phenotypes is unclear. Here, we show that the absence of EF-P affects the ability of B. subtilis to initiate sporulation by preventing normal expression of Spo0A, the key transcriptional regulator of this process. These data illustrate a mechanism that accounts for the sporulation delay and further suggest that cells are capable of sensing translation stress before committing to sporulation.

How to build a protoribosome: structural insights from the first protoribosome constructs that have proven to be catalytically active.

The modern ribosome catalyzes all coded protein synthesis in extant organisms. It is likely that its core structure is a direct descendant from the ribosome present in the last common ancestor (LCA). Hence, its earliest origins likely predate the LCA and therefore date further back in time. Of special interest is the pseudosymmetrical region (SymR) that lies deep within the large subunit (LSU) where the peptidyl transfer reaction takes place. It was previously proposed that two RNA oligomers, representing the P- and A-regions of extant ribosomes dimerized to create a pore-like structure, which hosted the necessary properties that facilitate peptide bond formation. However, recent experimental studies show that this may not be the case. Instead, several RNA constructs derived exclusively from the P-region were shown to form a homodimer capable of peptide bond synthesis. Of special interest will be the origin issues because the homodimer would have allowed a pre-LCA ribosome that was significantly smaller than previously proposed. For the A-region, the immediate issue will likely be its origin and whether it enhances ribosome performance. Here, we reanalyze the RNA/RNA interaction regions that most likely lead to SymR formation in light of these recent findings. Further, it has been suggested that the ability of these RNA constructs to dimerize and enhance peptide bond formation is sequence-dependent. We have analyzed the implications of sequence variations as parts of functional and nonfunctional constructs.

Peptide condensation and hydrolysis mechanisms from a proton-transfer network perspective.

Peptide bond formation is a fundamental organic chemical reaction; however, despite numerous recent reports, the computationally predicted barriers remain contradictory to experimental results. Incompleteness of the molecular mechanism for either the peptide bond formation or the reverse hydrolysis reactions is also highlighted by our lack of understanding of the seemingly equilibrium nature of the reaction that favors dipeptide formation over longer peptide chains under hydrothermal conditions. In this work, we first completed an assessment of levels of theory and evaluated chemical models that range from the neutral glycine condensation reaction in gas phase to explicitly solvated zwitterionic amino acids that are embedded in a polarizable continuum at neutral pH. Ultimately, we identified a six-step 'ping-pong' mechanism with the involvement of both zwitterions and neutral species. The carboxylate and amine end-groups of the diglycine intermediates play critical roles in the proton transfer and condensation processes. The experimental condensation barrier of 98 kJ mol-1 was approximated to be 118-129 kJ mol-1 at the MN15/def2TZVPP|SMD(water) level of theory for the rate determining step, when using the most complete model for the solvation environment. Condensed phase free energy correction employed to the rate limiting step reduced the barrier height to 106 kJ mol-1. These results have fundamental implications for understanding enzyme catalyzed peptide bond formation, peptide/protein stability, and "metabolism first" emergence of life scenarios.

Trimethylaluminum-mediated one-pot peptide elongation.

Efficient and straightforward peptide bond formation of N-, and C-terminal unprotected amino acids was successfully achieved by using trimethylaluminum. The coupling reaction was accomplished by pre-reaction of N-, and C-terminal unprotected amino acids and trimethylaluminum to form a five-membered ring that smoothly reacted with nucleophilic amino acid esters. This simple and highly efficient reaction system allows one-pot tripeptide synthesis without the need for expensive coupling reagents. Furthermore, peptide bond formation can be effectively achieved even for amino acids with bulky substituents at the side chain to afford the corresponding tripeptides in high yields in a one-pot manner. In addition, the reaction can be applied for further peptide elongation by the subsequent addition of amino acids and trimethylaluminum. We anticipate that this cost-effective, straightforward, and efficient protocol will be useful for the synthesis of a wide variety of peptides.

A simple geometrical model of the electrostatic environment around the catalytic center of the ribosome and its significance for the elongation cycle kinetics

The central function of the large subunit of the ribosome is to catalyze peptide bond formation. This biochemical reaction is conducted at the peptidyl transferase center (PTC). Experimental evidence shows that the catalytic activity is affected by the electrostatic environment around the peptidyl transferase center. Here, we set up a minimal geometrical model fitting the available x-ray solved structures of the ribonucleic cavity around the catalytic center of the large subunit of the ribosome. The purpose of this phenomenological model is to estimate quantitatively the electrostatic potential and electric field that are experienced during the peptidyl transfer reaction. At least two reasons motivate the need for developing this quantification. First, we inquire whether the electric field in this particular catalytic environment, made only of nucleic acids, is of the same order of magnitude as the one prevailing in catalytic centers of the proteic enzymes counterparts. Second, the protein synthesis rate is dependent on the nature of the amino acid sequentially incorporated in the nascent chain. The activation energy of the catalytic reaction and its detailed kinetics are shown to be dependent on the mechanical work exerted on the amino acids by the electric field, especially when one of the four charged amino acid residues (R, K, E, D) has previously been incorporated at the carboxy-terminal end of the peptidyl-tRNA. Physical values of the electric field provide quantitative knowledge of mechanical work, activation energy and rate of the peptide bond formation catalyzed by the ribosome. We show that our theoretical calculations are consistent with two independent sets of previously published experimental results. Experimental results for E.coli in the minimal case of the dipeptide bond formation when puromycin is used as the final amino acid acceptor strongly support our theoretically derived reaction time courses. Experimental Ribo-Seq results on E. coli and S. cerevisiae comparing the residence time distribution of ribosomes upon specific codons are also well accounted for by our theoretical calculations. The statistical queueing time theory was used to model the ribosome residence time per codon during nascent protein elongation and applied for the interpretation of the Ribo-Seq data. The hypo-exponential distribution fits the residence time observed distribution of the ribosome on a codon. An educated deconvolution of this distribution is used to estimate the rates of each elongation step in a codon specific manner. Our interpretation of all these results sheds light on the functional role of the electrostatic profile around the PTC and its impact on the ribosome elongation cycle.

Active ester-based peptide bond formation and its application in peptide synthesis

Active ester method is an efficient strategy to address the notorious racemization/epimerization issue of peptide bond formation. Herein, the pros and cons of using active esters for peptide synthesis were systematically summarized and analyzed.

Halogen-bonding-mediated synthesis of amides and peptides

We report the first methods for halogen-bonding-mediated formation of amide and peptide bonds. The methods can be used to synthesize not only amides but also dipeptides, tripeptides and pentapeptides in good to excellent yields without racemization.

Translation factor accelerating peptide bond formation on the ribosome: EF-P and eIF5A as entropic catalysts and a potential drug targets

Elongation factor P (EF-P) and its eukaryotic homolog eIF5A are auxiliary translation factors that facilitate peptide bond formation when several sequential proline (Pro) residues are incorporated into the nascent chain. EF-P and eIF5A bind to the exit (E) site of the ribosome and contribute to favorable entropy of the reaction by stabilizing tRNA binding in the peptidyl transferase center of the ribosome. In most organisms, EF-P and eIF5A carry a posttranslational modification that is crucial for catalysis. The chemical nature of the modification varies between different groups of bacteria and between pro- and eukaryotes, making the EF-P-modification enzymes promising targets for antibiotic development. In this review, we summarize our knowledge of the structure and function of EF-P and eIF5A, describe their modification enzymes, and present an approach for potential drug screening aimed at EarP, an enzyme that is essential for EF-P modification in several pathogenic bacteria.

Structural advances toward understanding the catalytic activity and conformational dynamics of modular nonribosomal peptide synthetases.

Covering: up to fall 2022.Nonribosomal peptide synthetases (NRPSs) are a family of modular, multidomain enzymes that catalyze the biosynthesis of important peptide natural products, including antibiotics, siderophores, and molecules with other biological activity. The NRPS architecture involves an assembly line strategy that tethers amino acid building blocks and the growing peptides to integrated carrier protein domains that migrate between different catalytic domains for peptide bond formation and other chemical modifications. Examination of the structures of individual domains and larger multidomain proteins has identified conserved conformational states within a single module that are adopted by NRPS modules to carry out a coordinated biosynthetic strategy that is shared by diverse systems. In contrast, interactions between modules are much more dynamic and do not yet suggest conserved conformational states between modules. Here we describe the structures of NRPS protein domains and modules and discuss the implications for future natural product discovery.

Effects of supplementation with different concentrations of L-citrulline on the plasma amino acid concentration, reproductive hormone concentrations, antioxidant capacity, and reproductive performance of Hu ewes

Context L-citrulline (L-Cit) does not degrade in the rumen and has the ability to form peptide bonds in the body; however, it does not participate in protein synthesis. Aims This study aimed to evaluate the effects of L-Cit on the reproductive performance of Hu ewes. Methods In total, 30 ewes were randomly categorised into five groups. The control group was fed with a basic diet, whereas the Experimental Groups I, II, III, and IV were provided feed supplemented with 5, 10, 15, and 20 g/day of L-Cit respectively. Blood samples of ewes were collected 4 h after feeding on Day 21 of the experiment and before feeding on Day 30. The optimal supplementary feeding dose was selected on the basis of blood biochemical indexes. Overall, ninety 2-year-old ewes were classified into two groups. The control group was fed with a basic diet and the experimental group was fed with a diet supplemented with 10 g/day of L-Cit. After 30 days of supplementary feeding, reproductive performance of ewes was determined. Key results The plasma concentrations of Cit, ornithine, and arginine in ewes increased linearly with an increase in the level of L-Cit supplementation. The plasma concentrations of gonadotropin-releasing hormone, luteinising hormone, and follicle-stimulating hormone in the experimental group increased significantly compared with those in the control group. The plasma total antioxidant capacity and catalase, superoxide dismutase, and glutathione peroxidase in the experimental group were significantly higher than those in the control group, whereas the concentrations of malondialdehyde in all experimental groups were significantly lower than those in the control group. The conception, lambing, and double lambing rates of the experimental group were increased by 28.76%, 15.90%, and 40.21% respectively. Conclusions Supplementation with different doses of L-Cit can improve the concentrations of some plasma amino acids and reproductive hormones as well as antioxidant capacity of ewes. Supplementary feeding with 10 g/day of L-Cit could increase the lambing and double lambing rates of ewes. Implication L-Cit can improve the reproductive performance of ewes.

Thiol functionalized activated carbon for gold thiosulfate recovery, an analysis of the interactions between gold and sulfur functions

Cyanide salts are currently used as leaching agents in gold extraction processes despite their toxicity. Thiosulfate is a greener choice, but its employment is constrained by the difficulty at recovering gold thiosulfate from the leachate using activated carbon, which in contrast, works well for gold-cyanide adsorption. Aiming to solve this problem, activated carbon has been covalently functionalized with thiol groups (-SH) through a peptide bond formation with cysteamine, as thiol-carrier molecule. The functionalized carbon accomplishes up to 100% Au recovery (with 2 and 25 ppm Au solution) in 8 h contact and its performance seems independent on the solution's pH. In-depth solid characterization techniques (XPS, TOF-SIMS, FTIR, PXRD, TEM, N₂ physisorption, elemental analysis) have been used to unravel the interactions between Au thiosulfate and thiol functions and two different gold adsorption mechanisms have been demonstrated: under acid conditions, the formation of Au1+- S chains has been proven. Under basic conditions, Au⁰ species have been detected in addition to Au1+- S chains. Elution of the gold loaded samples has been accomplished at a 71% level using Na₂S₂O₃ and NH₄OH as eluents.

Glycine to Oligoglycine via Sequential Trimetaphosphate Activation Steps in Drying Environments

Polyphosphate-mediated peptide bond formation is central to protein synthesis in modern organisms, but a simpler form of activation likely preceded the emergence of proteins and RNA. One suggested scenario involves trimetaphosphate (TP), an inorganic phosphate that promotes peptide condensation. Peptide bond formation can also be promoted by high pH and drying, but the interaction of these factors with TP has yet to be characterized kinetically. We studied the formation of glycine oligomers formed under initially alkaline conditions in the presence of TP during the process of drying. Oligopeptide products sampled over 24h were analyzed by functionalization and high-performance liquid chromatography with ultraviolet absorption (UV-HPLC). As they dried, two different pH-dependent mechanisms dominated during different stages of the process. The first mechanism occurs in alkaline solutions and activates monomer amino acids to form dimers while reducing the pH. Our results then become consistent with a second mechanism that proceeds at neutral pH and consumes dimers to form longer products. The possibility that a series of reactions might occur where the first reaction changes the environment to favor the second, and so on, may have broader implications for prebiotic polymerization. Studying how the environment changes during time-varying conditions, like drying, could help us understand how organic polymers formed during the origin of life.

Crystal structure of the AlbEF complex involved in subtilosin A biosynthesis

Subtilosin A is a sactipeptide bacteriocin produced by Bacillus subtilis strain 168, containing intramolecular thioether bonds and a head-to-tail macrocyclic peptide bond. Macrocyclization is presumably catalyzed by AlbE and AlbF proteins encoded by the subtilosin A biosynthesis gene cluster. However, the underlying mechanism of macrocyclization remains uncertain as the tertiary structures of the proteins are undetermined. Here, we report the crystal structures of AlbE and AlbF homologs in Quasibacillus thermotolerans, wherein the subtilosin biosynthesis gene cluster is highly conserved. Structural analysis and pull-down assays revealed that AlbE and AlbF form heterodimeric complexes. Although the AlbEF complex shows structural similarity to M16B family metalloproteases, the substrate-binding chamber is shallower and more open than the other M16B family proteins. The chamber surface showed electrostatic complementarity to the precursor of subtilosin. Our findings provide insights into the role of AlbEF in metalloprotease catalysis and macrocyclic peptide bond formation.

FRET Monitoring of a Nonribosomal Peptide Synthetase Elongation Module Reveals Carrier Protein Shuttling between Catalytic Domains.

Nonribosomal peptide synthetases (NRPSs) employ multiple domains, specifically arranged in modules, for the assembly-line biosynthesis of a plethora of bioactive peptides. It is poorly understood how catalysis is correlated with the domain interplay and associated conformational changes. We developed FRET sensors of an elongation module to study in solution the intramodular interactions of the peptidyl carrier protein (PCP) with adenylation (A) and condensation (C) domains. Backed by HDX-MS analysis, we discovered dynamic mixtures of conformations that undergo distinct population changes in favor of the PCP-A and PCP-C interactions upon completion of the adenylation and thiolation reactions, respectively. To probe this model we blocked PCP binding to the C domain by photocaging and triggered peptide bond formation with light. Changing intramodular domain affinities of the PCP appear to result in conformational shifts according to the logic of the templated assembly process.