Chemical Aminoacylation Research Articles

Orthogonality of Pyrrolysine tRNA in the Xenopus oocyte

Chemical aminoacylation of orthogonal tRNA allows for the genetic encoding of a wide range of synthetic amino acids without the need to evolve specific aminoacyl-tRNA synthetases. This method, when paired with protein expression in the Xenopus laevis oocyte expression system, can extract atomic scale functional data from a protein structure to advance the study of membrane proteins. The utility of the method depends on the orthogonality of the tRNA species used to deliver the amino acid. Here, we report that the pyrrolysyl tRNA (pylT) from Methanosarcina barkeri fusaro is orthogonal and highly competent for genetic code expansion experiments in the Xenopus oocyte. The data show that pylT is amendable to chemical acylation in vitro; it is then used to rescue a cytoplasmic site within a voltage-gated sodium channel. Further, the high fidelity of the pylT is demonstrated via encoding of lysine within the selectivity filter of the sodium channel, where sodium ion recognition by the distal amine of this side-chain is essential. Thus, pylT is an appropriate tRNA species for delivery of amino acids via nonsense suppression in the Xenopus oocyte. It may prove useful in experimental contexts wherein reacylation of suppressor tRNAs have been observed.

5(4H)-Oxazolones as Effective Aminoacylation Reagents for the 3′-Terminus of RNA

Nucleosides and methylated nucleotide models were used as substrates to identify pathways for the chemical aminoacylation of ribonucleic acids (RNA) as a prerequisite for the evolution of translation. A selective and comparatively efficient reaction of a 5(4 H )-oxazolone with the 2′- and 3′-OH of the ribonucleotide models was observed. Surprisingly, a similar reaction starting from an α-amino acid N -carboxyanhydride (NCA), selected as an acylating agent potentially leading to the unprotected ester required for translation, was not observed, which was confirmed using an acylated NCA equivalent. The reasons for this difference are analysed.

A Protein Expression System for Evaluating Cation-PI And PI-PI Interaction in Proteins

In proteins phenylalanine, tyrosine, and tryptophan can be involved in cation-pi and pi-pi interactions. The importance of these interactions for protein structure and functioning is well recognized but measuring their strength experimentally is problematic. The chemical aminoacylation strategy combined with the Xenopus oocyte expression system was developed by the Dougherty group at Caltech for the production of proteins labeled with fluorinated aromatic amino acids involved in cation-pi interactions. This elegant expression system only yields ng quantities of proteins, limiting the scope to channel-forming proteins analyzed via patch-clamp. We developed a protein expression platform for the evaluation of pi-pi and cation-pi interactions involving tryptophan. This Lactococcus lactis based expression system can produce mg amounts of a set of 6 proteins, each carrying a different electron density in the tryptophan side chain. It was used to express variants of the multidrug repressor protein LmrR. LmrR regulates the drug-induced expression of the ABC type multidrug efflux transporter LmrCD. LmrR was crystallized in its drug-free state and in complex with aromatic substrates showing each substrate is sandwiched between two tryptophan residues, suggesting pi-pi interactions are operative. The importance of pi-pi interactions for LmrR to bind aromatic substrates could now be quantitated using fluorescence, surface plasmon resonance, and X-ray crystallography. In conclusion, the newly developed expression system makes possible a systematic approach to study cation-pi and pi-pi interactions in proteins using a wide range of biophysical methods.

Facile synthesis of N-acyl-aminoacyl-pCpA for preparation of mischarged fully ribo tRNA.

Chemical synthesis of N-acyl-aminoacyl-pdCpA and its ligation to tRNA(minusCA) is widely used for the preparation of unnatural aminoacyl-tRNA substrates for ribosomal translation. However, the presence of the unnatural deoxyribose can decrease incorporation yield in translation and there is no straightforward method for chemical synthesis of the natural ribo version. Here, we show that pCpA is surprisingly stable to treatment with strong organic bases provided that anhydrous conditions are used. This allowed development of a facile method for chemical aminoacylation of pCpA. Preparative synthesis of pCpA was also simplified by using t-butyl-dithiomethyl protecting group methodology, and a more reliable pCpA postpurification treatment method was developed. Such aminoacyl-pCpA analogues ligated to tRNA(minusCA) transcripts are highly active in a purified translation system, demonstrating utility of our synthetic method.

ChemInform Abstract: In vivo Incorporation of Non‐Canonical Amino Acids by Using the Chemical Aminoacylation Strategy: A Broadly Applicable Mechanistic Tool

AbstractReview: [70 refs.

In vivo incorporation of non-canonical amino acids by using the chemical aminoacylation strategy: a broadly applicable mechanistic tool.

We describe a strategy for incorporating non-canonical amino acids site-specifically into proteins expressed in living cells, involving organic synthesis to chemically aminoacylate a suppressor tRNA, protein expression in Xenopus oocytes, and monitoring protein function, primarily by electrophysiology. With this protocol, a very wide range of non-canonical amino acids can be employed, allowing both systematic structure-function studies and the incorporation of reactive functionalities. Here, we present an overview of the methodology and examples meant to illustrate the versatility and power of the method as a tool for investigating protein structure and function.

Biosynthesis of proteins containing modified lysines and fluorescent labels using non-natural amino acid mutagenesis

The preparation of posttranslationally modified proteins is required to investigate the function and structure of modified proteins. However, homogeneously modified proteins are not easily isolated from natural sources or prepared using modification enzymes. Non-natural amino acid mutagenesis has enabled us to incorporate modified amino acids into specific positions of proteins in both cell-free and in-cell translation systems using tRNAs that are aminoacylated with modified amino acids. Here, we developed a method of double incorporation of modified amino acids and fluorescent non-natural amino acids in a quantitative, position-specific manner to obtain modified and fluorescently labeled proteins. To introduce methyllysine, dimethyllysine, trimethyllysine, and acetyllysine, frameshift and amber suppressor tRNAs aminoacylated with modified lysines were synthesized by chemical aminoacylation and supplied to an Escherichia coli cell-free translation system. The immunodetection of the translation products indicated that the modified lysines were incorporated into streptavidin and histone H3 in a quantitative, position-specific manner. Calmodulin derivatives containing a fluorescent non-natural amino acid at the N-terminal region and modified lysines at the Lys115 position were also synthesized, and their binding activity to a calmodulin-binding peptide was analyzed by fluorescence correlation spectroscopy. The results obtained here demonstrate that this method is useful in preparing and analyzing naturally occurring and non-natural modified proteins.

Chemical aminoacylation of tRNAs with fluorinated amino acids for in vitro protein mutagenesis.

This article describes the chemical aminoacylation of the yeast phenylalanine suppressor tRNA with a series of amino acids bearing fluorinated side chains via the hybrid dinucleotide pdCpA and ligation to the corresponding truncated tRNA species. Aminoacyl-tRNAs can be used to synthesize biologically relevant proteins which contain fluorinated amino acids at specific sites by means of a cell-free translation system. Such engineered proteins are expected to contribute to our understanding of discrete fluorines’ interaction with canonical amino acids in a native protein environment and to enable the design of fluorinated proteins with arbitrary desired properties.

Four-base codon-mediated saturation mutagenesis in a cell-free translation system

Saturation mutagenesis is a useful technique for the structural and functional analyses of proteins and for protein engineering. However, the extensive mutagenesis of genes and expression of mutated proteins are tedious and time-consuming. We have developed a simple and rapid method for the expression of mutated proteins with comprehensive single amino acid substitutions from single mutated genes having a four-base codon in a cell-free translation system. Twenty types of tRNA that were aminoacylated with one of the 20 proteinogenic amino acids and that contained a four-base anticodon were prepared by chemical aminoacylation. In the presence of one of the aminoacyl-tRNAs, a streptavidin mRNA with a four-base codon at the Tyr83 position was expressed in an Escherichia coli cell-free translation system. The N-terminus of the expressed proteins was fluorescently labeled using a fluorescent-labeled initiator Met-tRNA. Fluorescence imaging of an SDS-PAGE gel showed that all the amino acids are incorporated in response to the four-base codon; however, the incorporation efficiency was dependent on the structure of the side chains. Streptavidin mutants with comprehensive amino acid substitutions at the Tyr83, Arg84, and Tyr54 positions were used for analyzing their biotin-binding activity by dot blot analysis. These results demonstrate that this method is effective for the expression and analysis of mutated proteins with comprehensive amino acid substitutions at desired positions.

Chemical Aminoacylation of RNA by an Intermolecular Adenosine Transfer Reaction

Abstract RNA aminoacylation, based on an adenosine transfer mechanism, was achieved by the intermolecular transfer of phenylalaninyl adenosine from a donor probe to an acceptor RNA. This method can be applied to a wide variety of unnatural amino acids for tRNA aminoacylation.

Intermolecular transfer of aminoacylated adenosine towards chemical aminoacylation of tRNA

We synthesized two oligonucleotides: one is DNA conjugated with adenosine derivative at 5' terminus as a donor, another RNA phosphorothioated at 3' terminus as an acceptor. By hybridization of the two oligonuleotides, the adenosine derivative was transferred from the donor DNA to the acceptor RNA. In this study, O, O'-isopropylidene-protected adenosine derivative and aminoacylated adenosine were successfully transferred from the donor to acceptor RNA. By use of tRNA instead of the acceptor RNA, the chemical aminoacylation of tRNA by unnatural amino acid is expected.

Simple and quick chemical aminoacylation of tRNA in cationic micellar solution under ultrasonic agitation

A simple and quick method for direct aminoacylation of a tRNA with a non-natural amino acid was developed by using an N-protected amino acid cyanomethyl ester as a substrate solubilized in CTACl micelle under ultrasonic agitation.

In Situ Chemical Aminoacylation with Amino Acid Thioesters Linked to a Peptide Nucleic Acid

tRNA-specific chemical aminoacylation was achieved with nonnatural amino acids. A nonnatural amino acid was activated as a thioester derivative, and the latter was linked through a spacer to the N-terminal of a 9-mer peptide nucleic acid that is complementary to the 3'-terminal region of yeast phenylalanine tRNA. Efficient aminoacylation was observed when the amino acid thioester-spacer-PNA conjugate was mixed with the tRNA. The PNA-assisted aminoacylation was also successful in an Escherichia coli in vitro protein synthesizing system that contained an orthogonalized tRNA. The in situ aminoacylation/in vitro translation gave a mutant protein in which the nonnatural amino acid was incorporated into the position directed by a CGGG 4-base codon/anticodon pair.

Transformation of aminoacyl tRNAs for the in vitro selection of "drug-like" molecules.

Evolutionary approaches are regularly used to isolate single molecules with desired activities from large populations of nucleic acids (approximately 10(15)). Several methods have also been developed to generate libraries of mRNA-encoded peptides and proteins for the in vitro selection of functional polypeptides. In principal, such mRNA encoding systems could be used with libraries of nonbiological polymers if the ribosome can be directed to polymerize tRNAs carrying unnatural amino acids. The fundamental problem is that current chemical aminoacylation systems cannot easily produce sufficient amounts of the numerous misacylated tRNAs required to synthesize a complex library of encoded polymers. Here, we show that bulk-aminoacylated tRNA can be transformed into N-monomethylated aminoacyl tRNA and translated. Because poly-N-methyl peptide backbones are refractory to proteases and are membrane permeable, our method provides an uncomplicated means of evolving novel drug candidates.

Efficient Synthesis of Nonnatural Mutants in Escherichia coli S30 in vitro Protein Synthesizing System

Factors that affect the efficiency of in vitro synthesis of mutant proteins that contain nonnatural amino acids were investigated. The process of the nonnatural mutagenesis consists of chemical aminoacylation of a tRNA that contains a 4-base anticodon, followed by in vitro synthesis in the presence of an mRNA that contains the corresponding 4-base codon. Detailed studies on the time courses of the synthesis revealed two major factors that suppress the yield of nonnatural mutants compared with the wild-type protein. First, a cyclic tRNA that exists as a by-product of the chemical aminoacylation inhibits the protein synthesis. Second, the very short lifetime of a tRNA aminoacylated with a nonnatural amino acid limits the protein yield. As a simple and practical way of surmounting these factors, aminoacyl tRNA was added into the in vitro system at 5 min after the start of the synthesis. The addition increased the protein yield up to the level of conventional proteins in the in vitro system.

Incorporation of fluorescently labeled nonnatural amino acids into proteins in an E. coli in vitro translation system

Various nonnatural amino acids has been incorporated into proteins by using four-base codons in an E. coli in vitro translation system. Here, design and synthesis of novel fluorescently labeled nonnatural amino acids and their incorporation into proteins were investigated. Transfer RNAs that contained a CCCG anticodon and were aminoacylated with BODIPY FL-labeled amino acids were prepared by a chemical aminoacylation method, and added to an in vitro translation system in the presence of a streptavidin mRNA containing a CGGG codon. SDS-PAGE and Western blot analysis of the synthesized proteins indicate that BODIPY FL-labeled aminophenylalanine derivatives are efficiently incorporated into proteins through the four-base codon decoding.

Extension of Protein Functions by the Incorporation of Nonnatural Amino Acids

Abstract Site-specific mutagenesis of proteins was extended to incorporate nonnatural amino acids through in vitro cell-free translation or through in vivo translation in the living cells. To this end, a variety of nonnatural amino acids were synthesized and linked to tRNA by chemical aminoacylation. Further, unique codon/anticodon pairs were derived, such as the 4-base codon/anticodon pairs that are orthogonal to the conventional 3-base codon/anticodon pairs. By the extension of amino acids and the genetic codes, site-directed incorporation of specialty amino acids into a variety of proteins has been achieved. Amino acids that carry large aromatic side groups with straight configurations, such as 2-anthrylalanine and 2-anthraquinonylalanine were successfully introduced into proteins and the CGGG or AGGU 4-base codons were used to assign their positions. Amino acids carrying photocleavable side groups, a spin probe, fluorescent probes, and other functional groups have been introduced into proteins. The specialty proteins were shown to be powerful tools for biochemical research and are expected to be novel molecules with designed arrangements of functional groups on the protein framework.

A general and efficient route for chemical aminoacylation of transfer RNAs

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTA general and efficient route for chemical aminoacylation of transfer RNAsStephanie A. Robertson, Jonathan A. Ellman, and Peter G. SchultzCite this: J. Am. Chem. Soc. 1991, 113, 7, 2722–2729Publication Date (Print):March 1, 1991Publication History Published online1 May 2002Published inissue 1 March 1991https://doi.org/10.1021/ja00007a055RIGHTS & PERMISSIONSArticle Views1914Altmetric-Citations169LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

The use of 5'-phospho-2 deoxyribocytidylylriboadenosine as a facile route to chemical aminoacylation of tRNA.

Methodology is described for the synthesis and chemical aminoacylation of the hybrid dinucleotide 5'-phospho-2'-deoxyribocytidylylriboadenosine (pdCpA). Ligation of aminoacylated pdCpA to a truncated amber suppressor tRNACUA (-CA) using T4 RNA ligase generates an aminoacylated suppressor tRNA which can be used for site-specific incorporation of unnatural amino acids into proteins. Both the ligation and in vitro suppression efficiencies are the same when either pCpA or pdCpA is used. The use of deoxycytidine simplifies the chemistry involved in the synthesis of the dinucleotide pCpA. In addition, these results demonstrate that ribocytidine is not required for recognition of the aminoacylated tRNA during protein synthesis.

Ribosome-catalyzed formation of an abnormal peptide analogue.

The peptidyl-tRNA analogue N-(chloroacetyl)phenylalanyl-tRNAPhe was prepared by chemical aminoacylation and prebound to the P site of Escherichia coli ribosomes in response to poly(uridylic acid). Admixture of phenylalanyl-tRNAPhe to the A site resulted in the formation of two "dipeptides", one of which was formed by displacement of chloride ion from the peptidyl-tRNA. This constitutes the first example of ribosome-mediated formation of a peptide of altered connectivity and suggests a need for revision of the current model of peptide bond formation. Also suggested by the present finding is the feasibility of utilizing tRNAs to prepare polypeptides of altered connectivity in an in vitro protein biosynthesizing system.