Protein Expression with Biosynthesized Noncanonical Amino Acids.

Natural proteins are normally made by 20 canonical amino acids. Genetic code expansion (GCE) enables incorporation of diverse chemically synthesized noncanonical amino acids (ncAAs) by orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs using nonsense codons, which could significantly expand new functionalities of proteins in both scientific and biomedical applications. Here, by hijacking the cysteine biosynthetic enzymes, we describe a method combining amino acid biosynthesis and GCE to introduce around 50 structurally novel ncAAs into proteins by supplementation of commercially available aromatic thiol precursors, thus eliminating the need to chemically synthesize these ncAAs. A screening method is also provided for improving the incorporation efficiency of a particular ncAA. Furthermore, we demonstrate bioorthogonal groups, such as azide and ketone, that are compatible with our system and can be easily introduced into protein for subsequent site-specific labeling.

Adaptation to Overflow Metabolism by Mutations That Impair tRNA Modification in Experimentally Evolved Bacteria.

When microbes grow in foreign nutritional environments, selection may enrich mutations in unexpected pathways connecting growth and homeostasis. An evolution experiment designed to identify beneficial mutations in Burkholderia cenocepacia captured six independent nonsynonymous substitutions in the essential gene tilS, which modifies tRNAIle2 by adding a lysine to the anticodon for faithful AUA recognition. Further, five additional mutants acquired mutations in tRNAIle2, which strongly suggests that disrupting the TilS-tRNAIle2 interaction was subject to strong positive selection. Mutated TilS incurred greatly reduced enzymatic function but retained capacity for tRNAIle2 binding. However, both mutant sets outcompeted the wild type by decreasing the lag phase duration by ~3.5 h. We hypothesized that lysine demand could underlie fitness in the experimental conditions. As predicted, supplemental lysine complemented the ancestral fitness deficit, but so did the additions of several other amino acids. Mutant fitness advantages were also specific to rapid growth on galactose using oxidative overflow metabolism that generates redox imbalance, not resources favoring more balanced metabolism. Remarkably, 13 tilS mutations also evolved in the long-term evolution experiment with Escherichia coli, including four fixed mutations. These results suggest that TilS or unknown binding partners contribute to improved growth under conditions of rapid sugar oxidation at the predicted expense of translational accuracy. IMPORTANCE There is growing evidence that the fundamental components of protein translation can play multiple roles in maintaining cellular homeostasis. Enzymes that interact with transfer RNAs not only ensure faithful decoding of the genetic code but also help signal the metabolic state by reacting to imbalances in essential building blocks like free amino acids and cofactors. Here, we present evidence of a secondary function for the essential enzyme TilS, whose only prior known function is to modify tRNAIle(CAU) to ensure accurate translation. Multiple nonsynonymous substitutions in tilS, as well as its cognate tRNA, were selected in evolution experiments favoring rapid, redox-imbalanced growth. These mutations alone decreased lag phase and created a competitive advantage, but at the expense of most primary enzyme function. These results imply that TilS interacts with other factors related to the timing of exponential growth and that tRNA-modifying enzymes may serve multiple roles in monitoring metabolic health.

Directed-evolution of translation system for efficient unnatural amino acids incorporation and generalizable synthetic auxotroph construction.

Site-specific incorporation of unnatural amino acids (UAAs) with similar incorporation efficiency to that of natural amino acids (NAAs) and low background activity is extremely valuable for efficient synthesis of proteins with diverse new chemical functions and design of various synthetic auxotrophs. However, such efficient translation systems remain largely unknown in the literature. Here, we describe engineered chimeric phenylalanine systems that dramatically increase the yield of proteins bearing UAAs, through systematic engineering of the aminoacyl-tRNA synthetase and its respective cognate tRNA. These engineered synthetase/tRNA pairs allow single-site and multi-site incorporation of UAAs with efficiencies similar to those of NAAs and high fidelity. In addition, using the evolved chimeric phenylalanine system, we construct a series of E. coli strains whose growth is strictly dependent on exogenously supplied of UAAs. We further show that synthetic auxotrophic cells can grow robustly in living mice when UAAs are supplemented.

Multiplex suppression of four quadruplet codons via tRNA directed evolution.

Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon-anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system.

YY1 and RTCB in mouse uterine decidualization and embryo implantation.

As a multifunctional transcription factor, YY1 regulates the expression of many genes essential for early embryonic development. RTCB is an RNA ligase that plays a role in tRNA maturation and Xbp1 mRNA splicing. YY1 can bind in vitro to the response element in the proximal promoter of Rtcb and regulate Rtcb promoter activity. However, the in vivo regulation and whether these two genes are involved in the mother-fetal dialogue during early pregnancy remain unclear. In this study, we validated that YY1 bound in vivo to the proximal promoter of Rtcb in mouse uterus of early pregnancy. Moreover, via building a variety of animal models, our study suggested that both YY1 and RTCB might play a role in mouse uterus decidualization and embryo implantation during early pregnancy.

Amino acid activation analysis of primitive aminoacyl-tRNA synthetases encoded by both strands of a single gene using the malachite green assay.

The Rodin-Ohno hypothesis postulates that two classes of aminoacyl-tRNA synthetases were encoded complementary to double-stranded DNA. Particularly, Geobacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS, belonging to class I) and Escherichia coli histidyl-tRNA synthetase (HisRS, belonging to class II) show high complementarity of the middle base of the codons in the mRNA sequence encoding each ATP binding site. Here, for the reported 46-residue peptides designed from the three-dimensional structures of TrpRS and HisRS, amino acid activation analysis was performed using the malachite green assay, which detects the pyrophosphate departing from ATP in the forward reaction of the first step of tRNA aminoacylation. A maltose-binding protein fusion with the 46 residues of TrpRS (TrpRS46mer) exhibited high activation capacity for several amino acids in the presence of ATP and amino acids, but the activity of an alanine substitution mutant of the first histidine in the HIGH motif (TrpRS46merH15A) was largely reduced. In contrast, pyrophosphate release by HisRS46mer in the histidine activation step was lower than that in the case of TrpRS46mer. Both HisRS46mer and the alanine mutant at the 113th arginine (HisRS46merR113A) showed slightly higher levels of pyrophosphate release than the maltose-binding protein alone. These results do not rule out the Rodin-Ohno hypothesis, but may suggest the necessity of establishing unique evolutionary models from different perspectives.

Site-Specific Incorporation of Two ncAAs for Two-Color Bioorthogonal Labeling and Crosslinking of Proteins on Live Mammalian Cells.

The pyrrolysyl-tRNA/pyrrolysyl-tRNA synthetase (PylT/RS) pair from the archaeon Methanosarcina mazei (Mma) is widely used in protein engineering to site-specifically introduce noncanonical amino acids (ncAAs) through nonsense codon suppression. Here, we engineer the PylT/RS pair encoded by Methanogenic archaeon ISO4-G1 (G1) to be orthogonal to Mma PylT/RS and alter the G1 PylRS active site to accept a complementary ncAA spectrum. We combine the resulting mutual orthogonal pairs for site-specific dual ncAA incorporation of two lysine analogs with high selectivity and efficiency. Demonstrating the robustness of the system, we incorporate two ncAAs with compatible bioorthogonal reactivity into a Notch receptor, as well as a G protein-coupled receptor. We show that selective and site-specific incorporation of two ncAAs allows for two-color bioorthogonal labeling as well as chemical-controlled crosslinking of surface proteins on live mammalian cells.

Codon Directional Asymmetry Suggests Swapped Prebiotic 1st and 2nd Codon Positions.

Background: Codon directional asymmetry (CDA) classifies the 64 codons into palindromes (XYX, CDA = 0), and 5'- and 3'-dominant (YXX and XXY, CDA < 0 and CDA > 0, respectively). Previously, CDA was defined by the purine/pyrimidine divide (A,G/C,T), where X is either a purine or a pyrimidine. For the remaining codons with undefined CDA, CDA was defined by the 5' or 3' nucleotide complementary to Y. This CDA correlates with cognate amino acid tRNA synthetase classes, antiparallel beta sheet conformation index and the evolutionary order defined by the self-referential genetic code evolution model (CDA < 0: class I, high beta sheet index, late genetic code inclusion). Methods: We explore associations of CDAs defined by nucleotide classifications according to complementarity strengths (A:T, weak; C:G, strong) and keto-enol/amino-imino groupings (G,T/A,C), also after swapping 1st and 2nd codon positions with amino acid physicochemical and structural properties. Results: Here, analyses show that for the eight codons whose purine/pyrimidine-based CDA requires using the rule of complementarity with the midposition, using weak interactions to define CDA instead of complementarity increases associations with tRNA synthetase classes, antiparallel beta sheet index and genetic code evolutionary order. CDA defined by keto-enol/amino-imino groups, 1st and 2nd codon positions swapped, correlates with amino acid parallel beta sheet formation indices and Doolittle's hydropathicities. Conclusions: Results suggest (a) prebiotic swaps from N2N1N3 to N1N2N3 codon structures, (b) that tRNA-mediated translation replaced direct codon-amino acid interactions, and (c) links between codon structures and cognate amino acid properties.

Glutaminyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization, structure, and development as a screening platform.

Pseudomonas aeruginosa has a high potential for developing resistance to multiple antibiotics. The gene (glnS) encoding glutaminyl-tRNA synthetase (GlnRS) from P. aeruginosa was cloned and the resulting protein characterized. GlnRS was kinetically evaluated and the KM and kcatobs , governing interactions with tRNA, were 1.0 µM and 0.15 s-1 , respectively. The crystal structure of the α2 form of P. aeruginosa GlnRS was solved to 1.9 Å resolution. The amino acid sequence and structure of P. aeruginosa GlnRS were analyzed and compared to that of GlnRS from Escherichia coli. Amino acids that interact with ATP, glutamine, and tRNA are well conserved and structure overlays indicate that both GlnRS proteins conform to a similar three-dimensional structure. GlnRS was developed into a screening platform using scintillation proximity assay technology and used to screen ~2,000 chemical compounds. Three inhibitory compounds were identified and analyzed for enzymatic inhibition as well as minimum inhibitory concentrations against clinically relevant bacterial strains. Two of the compounds, BM02E04 and BM04H03, were selected for further studies. These compounds displayed broad-spectrum antibacterial activity and exhibited moderate inhibitory activity against mutant efflux deficient strains of P. aeruginosa and E. coli. Growth of wild-type strains was unaffected, indicating that efflux was likely responsible for the lack of sensitivity. The global mode of action was determined using time-kill kinetics. BM04H03 did not inhibit the growth of human cell cultures at any concentration and BM02E04 only inhibit cultures at the highest concentration tested (400 µg/ml). In conclusion, GlnRS from P. aeruginosa is shown to have a structure similar to that of E. coli GlnRS and two natural product compounds were identified as inhibitors of P. aeruginosa GlnRS with the potential for utility as lead candidates in antibacterial drug development in a time of increased antibiotic resistance.

Aminoacyl-tRNA synthetases as therapeutic targets.

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes for protein synthesis with evolutionarily conserved enzymatic mechanisms. Despite their similarity across organisms, scientists have been able to generate effective anti-infective agents based on the structural differences in the catalytic clefts of ARSs from pathogens and humans. However, recent genomic, proteomic and functionomic advances have unveiled unexpected disease-associated mutations and altered expression, secretion and interactions in human ARSs, revealing hidden biological functions beyond their catalytic roles in protein synthesis. These studies have also brought to light their potential as a rich and unexplored source for new therapeutic targets and agents through multiple avenues, including direct targeting of the catalytic sites, controlling disease-associated protein-protein interactions and developing novel biologics from the secreted ARS proteins or their parts. This Review addresses the emerging biology and therapeutic applications of human ARSs in diseases including autoimmune and rare diseases, and cancer.

Site-Specific Labeling of F-18 Proteins Using a Supplemented Cell-Free Protein Synthesis System and O-2-[18F]Fluoroethyl-L-Tyrosine: [18F]FET-HER2 Affibody Molecule.

PURPOSE: Although a preparation method for F-18-labeled proteins that used a cell-free translation system and 4-[18F]fluoro-L-proline instead of L-proline has been reported, its introduction depends on amino acid sequences of target proteins. The purpose of the study was to propose site-specific labeling method of F-18 by using cell-free translation systems supplemented with an engineered orthogonal aminoacyl-tRNA synthetase derived from Methanocaldococcus jannaschii (pCNF-RS)/suppressor tRNA (tRNACUAopt) pair, O-2-[18F]fluoroethyl-L-tyrosine ([18F]FET), and template DNA inserted with an amber codon. PROCEDURES: [18F]FET was prepared from the corresponding precursor and determined whether [18F]FET could be incorporated into an affibody molecule for human epidermal growth factor receptor type 2 (HER2; ZHER2:342) as the 21st amino acid used with the pCNF-RS-tRNACUAopt pair and template DNA inserted with an amber codon in a cell-free translation system. Using SKOV-3 cells, we performed an in vitro binding assay of [18F]FET-ZHER2:342. Furthermore, in vivo positron emission tomography (PET) imaging in SKOV-3 xenograft-bearing mice was performed after the intravenous administration of [18F]FET-ZHER2:342. RESULTS: [18F]FET was successfully incorporated into proteins by using commercially available cell-free protein synthesis reagents with a pCNF-RS-tRNACUAopt pair and template DNA of the desired proteins inserted with an amber codon. The mean radiochemical yield (non-decay-corrected) of [18F]FET-ZHER2:342 was 6.5 ± 4.1 %. An in vitro cell binding assay revealed that SKOV-3 cells-bound [18F]FET-ZHER2:342 expressed HER2. The in vivo PET imaging in SKOV-3 xenograft-bearing mice revealed that [18F]FET-ZHER2:342 accumulated in SKOV-3 xenografts. CONCLUSION: The method proposed in this study might be useful for preparing proteins with F-18 and molecular imaging in the preclinical development.

Aminoacyl-tRNA synthetases, therapeutic targets for infectious diseases.

Despite remarkable advances in medical science, infection-associated diseases remain among the leading causes of death worldwide. There is a great deal of interest and concern at the rate at which new pathogens are emerging and causing significant human health problems. Expanding our understanding of how cells regulate signaling networks to defend against invaders and retain cell homeostasis will reveal promising strategies against infection. It has taken scientists decades to appreciate that eukaryotic aminoacyl-tRNA synthetases (ARSs) play a role as global cell signaling mediators to regulate cell homeostasis, beyond their intrinsic function as protein synthesis enzymes. Recent discoveries revealed that ubiquitously expressed standby cytoplasmic ARSs sense and respond to danger signals and regulate immunity against infections, indicating their potential as therapeutic targets for infectious diseases. In this review, we discuss ARS-mediated anti-infectious signaling and the emerging role of ARSs in antimicrobial immunity. In contrast to their ability to defend against infection, host ARSs are inevitably co-opted by viruses for survival and propagation. We therefore provide a brief overview of the communication between viruses and the ARS system. Finally, we discuss encouraging new approaches to develop ARSs as therapeutics for infectious diseases.

Backbone Brackets and Arginine Tweezers delineate Class I and Class II aminoacyl tRNA synthetases.

The origin of the machinery that realizes protein biosynthesis in all organisms is still unclear. One key component of this machinery are aminoacyl tRNA synthetases (aaRS), which ligate tRNAs to amino acids while consuming ATP. Sequence analyses revealed that these enzymes can be divided into two complementary classes. Both classes differ significantly on a sequence and structural level, feature different reaction mechanisms, and occur in diverse oligomerization states. The one unifying aspect of both classes is their function of binding ATP. We identified Backbone Brackets and Arginine Tweezers as most compact ATP binding motifs characteristic for each Class. Geometric analysis shows a structural rearrangement of the Backbone Brackets upon ATP binding, indicating a general mechanism of all Class I structures. Regarding the origin of aaRS, the Rodin-Ohno hypothesis states that the peculiar nature of the two aaRS classes is the result of their primordial forms, called Protozymes, being encoded on opposite strands of the same gene. Backbone Brackets and Arginine Tweezers were traced back to the proposed Protozymes and their more efficient successors, the Urzymes. Both structural motifs can be observed as pairs of residues in contemporary structures and it seems that the time of their addition, indicated by their placement in the ancient aaRS, coincides with the evolutionary trace of Proto- and Urzymes.

Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics.

Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate L-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.

Comparative analysis of polyspecificity of the endogenous tRNA synthetase of different expression host towards photocrosslinking amino acids using an in silico approach.

Photo-induced covalent crosslinking has emerged as the powerful strategy for analyzing and characterizing the protein-protein interaction and mapping protein 3D conformations. In the last decades, a number of photocrosslinking amino acids have been reported but only a few have been efficiently utilized for photocrosslinking purposes. Recently, incorporation of diazirine containing photoactivatable analogs such as photo-methionine, photo-leucine, photo-isoleucine and photo-lysine into target proteins were accomplished in live cells (Human A549cells, HEK 293) by depleting corresponding natural amino acid and supplementing these analogs in the medium. Likewise, incorporation of photo-methionine and photo-leucine is also reported in E. coli. Incorporation of these unnatural amino acids were demonstrated only in a limited number species, thereby conventional methods have been utilized for the protein-protein interaction study in other species. With this in mind, we studied in silico analysis of polyspecificity of four endogenous tRNA synthetases (LeuRS, IleRS, MetRS, and LysRS) from six different species such as Escherichia coli, Pseudomonas fluorescens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Aspergillus oryzae and Homo sapiens towards its photocrosslinking amino acids. In addition, here we describe the active site similarity of different protein bio-factories. Based on the active site similarity and similar binding mode, we predicted that the endogenous tRNA synthetases of all the species are reactive to corresponding photoactivatable analogs. This is the first in silico study to demonstrate that the photocrosslinking unnatural amino acids are recognized by the endogenous tRNA synthetases of different protein expression biofactories.

Activation of the Amino Acid Response Pathway Blunts the Effects of Cardiac Stress.

BACKGROUND: The amino acid response (AAR) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti-inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl-tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure. METHODS AND RESULTS: Consistent with its ability to inhibit prolyl-tRNA synthetase, halofuginone elicited a general control nonderepressible 2-dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l-proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II/phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2-dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell-derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin-1-mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/eIF2α-dependent manner. CONCLUSIONS: Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.

A Novel Organic Selenium Compound Exerts Unique Regulation of Selenium Speciation, Selenogenome, and Selenoproteins in Broiler Chicks.

Background: A new organic selenium compound, 2-hydroxy-4-methylselenobutanoic acid (SeO), displayed a greater bioavailability than sodium selenite (SeNa) or seleno-yeast (SeY) in several species.Objective: This study sought to determine the regulation of the speciation of selenium, expression of selenogenome and selenocysteine biosynthesis and degradation-related genes, and production of selenoproteins by the 3 forms of selenium in the tissues of broiler chicks.Methods: Day-old male chicks (n = 6 cages/diet, 6 chicks/cage) were fed a selenium-deficient, corn and soy-based diet [base diet (BD), 0.05 mg Se/kg] or the BD + SeNa, SeY, or SeO at 0.2 mg Se/kg for 6 wk. Plasma, livers, and pectoral and thigh muscles were collected at weeks 3 and 6 to assay for total selenium, selenomethionine, selenocysteine, redox status, and selected genes, proteins, and enzymes.Results: Although both SeY and SeO produced greater concentrations (P < 0.05) of total selenium (20-172%) and of selenomethionine (≤15-fold) in the liver, pectoral muscle, and thigh than those of SeNa, SeO further raised (P < 0.05) these concentrations by 13-37% and 43-87%, respectively, compared with SeY. Compared with the BD, only SeO enhanced (P < 0.05) the mRNA of selenoprotein (Seleno) s and methionine sulfoxide reductase B1 (Msrb1) in the liver and thigh (62-98%) and thioredoxin reductase (TXRND) activity in the pectoral and thigh muscles (20-37%) at week 3. Furthermore, SeO increased (P < 0.05) the expression of glutathione peroxidase (Gpx) 3, GPX4, SELENOP, and SELENOU relative to the SeNa group by 26-207%, and the expression of Selenop, O-phosphoseryl-transfer RNA (tRNA):selenocysteinyl-tRNA synthase, GPX4, and SELENOP relative to the SeY group by 23-55% in various tissues.Conclusions: Compared with SeNa or SeY, SeO demonstrated a unique ability to enrich selenomethionine and total selenium depositions, to induce the early expression of Selenos and Mrsb1 mRNA and TXRND activity, and to enhance the protein production of GPX4, SELENOP, and SELENOU in the tissues of chicks.

Exploring dual inhibitory role of febrifugine analogues against Plasmodium utilizing structure-based virtual screening and molecular dynamic simulation.

Malaria is an endemic disease caused by the protozoan parasite Plasomodium falciparum. Febrifugine analogues are natural compound obtained from the traditional Chinese herbs have shown significant antimalarial and anticancerous efficacy in experimental model. Development of resistance against the existing antimalarial drug has alarmed the scientific innovators to find a potential antimalarial molecule which can be further used by endemic countries for the elimination of this disease. In this study, structure-based virtual screening and molecular dynamics (MD) base approaches were used to generate potential antimalarial compound against plasmepsin II and prolyl-tRNA synthetase of Plasmodium. Here, we have docked series of febrifugine analogues (n = 11,395) against plasmepsin II in three different docking modes and then it was compared with previously reported target prolyl-tRNA synthetase. Extra precision docking resulted into 235 ligands having better docking score were subject for QikProp analysis. Better ligands (n = 39) obtained from QikProp analysis were subject for ADMET prediction and docking protocol validation through the estimation of receiver operator characteristics. In the later stage, 24 ligands obtained from ADMET study were subject for the estimation of binding energy through MM-GBSA and same were also docked against prolyl-tRNA synthetase to get compounds with dual inhibitor role. Finally, MD simulation and 2D fingerprint MACCS study of two best ligands have shown significant interaction with plasmepsin II and homology against known active ligand with noteworthy MACCS index, respectively. This study concludes that FA12 could be potential drug candidate to fight against Plasmodium falciparum parasites.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System.

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Functional Class I and II Amino Acid-activating Enzymes Can Be Coded by Opposite Strands of the Same Gene.

Aminoacyl-tRNA synthetases (aaRS) catalyze both chemical steps that translate the universal genetic code. Rodin and Ohno offered an explanation for the existence of two aaRS classes, observing that codons for the most highly conserved Class I active-site residues are anticodons for corresponding Class II active-site residues. They proposed that the two classes arose simultaneously, by translation of opposite strands from the same gene. We have characterized wild-type 46-residue peptides containing ATP-binding sites of Class I and II synthetases and those coded by a gene designed by Rosetta to encode the corresponding peptides on opposite strands. Catalysis by WT and designed peptides is saturable, and the designed peptides are sensitive to active-site residue mutation. All have comparable apparent second-order rate constants 2.9-7.0E-3 M(-1) s(-1) or ∼750,000-1,300,000 times the uncatalyzed rate. The activities of the two complementary peptides demonstrate that the unique information in a gene can have two functional interpretations, one from each complementary strand. The peptides contain phylogenetic signatures of longer, more sophisticated catalysts we call Urzymes and are short enough to bridge the gap between them and simpler uncoded peptides. Thus, they directly substantiate the sense/antisense coding ancestry of Class I and II aaRS. Furthermore, designed 46-mers achieve similar catalytic proficiency to wild-type 46-mers by significant increases in both kcat and Km values, supporting suggestions that the earliest peptide catalysts activated ATP for biosynthetic purposes.