Elongation Factor P Modulates the Incorporation of Structurally Diverse Noncanonical Amino Acids into Escherichia coli Dihydrofolate Reductase.

The introduction of noncanonical amino acids into proteins and peptides has been of great interest for many years and has facilitated the detailed study of peptide/protein structure and mechanism. In addition to numerous nonproteinogenic α-l-amino acids, bacterial ribosome modification has provided the wherewithal to enable the synthesis of peptides and proteins with a much greater range of structural diversity, as has the use of endogenous bacterial proteins in reconstituted protein synthesizing systems. In a recent report, elongation factor P (EF-P), putatively essential for enabling the incorporation of contiguous proline residues into proteins, was shown to facilitate the introduction of an N-methylated amino acid in addition to proline. This finding prompted us to investigate the properties of this protein factor with a broad variety of structurally diverse amino acid analogues using an optimized suppressor tRNAPro that we designed. While these analogues can generally be incorporated into proteins only in systems containing modified ribosomes specifically selected for their incorporation, we found that EF-P could significantly enhance their incorporation into model protein dihydrofolate reductase using wild-type ribosomes. Plausibly, the increased yields observed in the presence of structurally diverse amino acid analogues may result from the formation of a stabilized ribosomal complex in the presence of EF-P that provides more favorable conditions for peptide bond formation. This finding should enable the facile incorporation of a much broader structural variety of amino acid analogues into proteins and peptides using native ribosomes.

Lasso-grafting of macrocyclic peptide pharmacophores yields multi-functional proteins.

Protein engineering has great potential for devising multifunctional recombinant proteins to serve as next-generation protein therapeutics, but it often requires drastic modifications of the parental protein scaffolds e.g., additional domains at the N/C-terminus or replacement of a domain by another. A discovery platform system, called RaPID (Random non-standard Peptides Integrated Discovery) system, has enabled rapid discovery of small de novo macrocyclic peptides that bind a target protein with high binding specificity and affinity. Capitalizing on the optimized binding properties of the RaPID-derived peptides, here we show that RaPID-derived pharmacophore sequences can be readily implanted into surface-exposed loops on recombinant proteins and maintain both the parental peptide binding function(s) and the host protein function. We refer to this protein engineering method as lasso-grafting and demonstrate that it can endow specific binding capacity toward various receptors into a diverse set of scaffolds that includes IgG, serum albumin, and even capsid proteins of adeno-associated virus, enabling us to rapidly formulate and produce bi-, tri-, and even tetra-specific binder molecules.

Ribosomal Formation of Thioamide Bonds in Polypeptide Synthesis.

It has been well established that the ribosome can accept various nucleophiles on the Xacyl-tRNA in the A site during elongation, where X can be amino, N-alkyl-amino, hydroxy, and thiol groups. However, it remains elusive that the ribosome is able to accept an electrophile in the P site other than the carboxyl group during elongation. Here we report ribosomal formation of a thioamide bond in the mRNA-dependent polypeptide synthesis. In this study, amino(carbothio)acyl-tRNA was prepared by flexizyme and used for the expression of peptides containing a thioamide bond in the nascent peptide chain. We give strong evidence that the thioamide-peptide was formed but accompanied by the amide counterpart due to rapid carbo(S-to-O) exchange during the preparation of amino(carbothio)acyl-tRNA. We also demonstrate the ribosomal formation of thioamide and N-methyl-thioamide bonds in linear as well as macrocyclic peptide scaffolds in the mRNA-dependent manner, showing its potential for applications in peptide-based drug discovery and studying peptide/protein structure and function.

Incorporation of Phosphorylated Tyrosine into Proteins: In Vitro Translation and Study of Phosphorylated IκB-α and Its Interaction with NF-κB.

Phosphorylated proteins play important roles in the regulation of many different cell networks. However, unlike the preparation of proteins containing unmodified proteinogenic amino acids, which can be altered readily by site-directed mutagenesis and expressed in vitro and in vivo, the preparation of proteins phosphorylated at predetermined sites cannot be done easily and in acceptable yields. To enable the synthesis of phosphorylated proteins for in vitro studies, we have explored the use of phosphorylated amino acids in which the phosphate moiety bears a chemical protecting group, thus eliminating the negative charges that have been shown to have a negative effect on protein translation. Bis-o-nitrobenzyl protection of tyrosine phosphate enabled its incorporation into DHFR and IκB-α using wild-type ribosomes, and the elaborated proteins could subsequently be deprotected by photolysis. Also investigated in parallel was the re-engineering of the 23S rRNA of Escherichia coli, guided by the use of a phosphorylated puromycin, to identify modified ribosomes capable of incorporating unprotected phosphotyrosine into proteins from a phosphotyrosyl-tRNACUA by UAG codon suppression during in vitro translation. Selection of a library of modified ribosomal clones with phosphorylated puromycin identified six modified ribosome variants having mutations in nucleotides 2600-2605 of 23S rRNA; these had enhanced sensitivity to the phosphorylated puromycin. The six clones demonstrated some sequence homology in the region 2600-2605 and incorporated unprotected phosphotyrosine into IκB-α using a modified gene having a TAG codon in the position corresponding to amino acid 42 of the protein. The purified phosphorylated protein bound to a phosphotyrosine specific antibody and permitted NF-κB binding to a DNA duplex sequence corresponding to its binding site in the IL-2 gene promoter. Unexpectedly, phosphorylated IκB-α also mediated the exchange of exogenous DNA into an NF-κB-cellular DNA complex isolated from the nucleus of activated Jurkat cells.

Ribosome-mediated synthesis of natural product-like peptides via cell-free translation.

Peptide natural products (PNPs) represent a unique class of compounds known for their fascinating structural motifs with important biological activities. Lately, PNPs have garnered a lot of interest for their application in drug discovery. Nevertheless, lack of diversity oriented synthetic/biosynthetic platforms to generate large natural product-like libraries has limited their development as peptide therapeutics. The promiscuity of cell-free translation has allowed for the synthesis of artificial PNPs having complex structural features. Modified cell-free translation systems coupled with the display technologies have generated diverse natural product-like peptide libraries and led to the discovery of several biologically active molecules. Such technologies have drastically decreased the time to obtain peptide drug leads and therefore, revolutionized the field of peptide drug discovery. In this account, we review recent developments in the synthesis of natural product-like peptides via cell-free translation.

Synthesis of fluorescent dipeptidomimetics and their ribosomal incorporation into green fluorescent protein.

The synthesis and incorporation into position 66 of green fluorescent protein (GFP) by in vitro protein translation of novel oxazole and thiazole based dipeptidomimetics are described. The compounds may be regarded as GFP chromophore analogues, and are strongly fluorescent. An α-amido-ß-ketoester intermediate was obtained via bisacylation of a protected glycine. The intermediate underwent dehydrative cyclization to afford the 1,3-oxazole and was treated with Lawesson's reagent to furnish the 1,3-thiazole. When these fluorophores were introduced into position 66 of GFP in place of Tyr66, the resulting GFP analogues exhibited fluorescence emission several-fold greater than wild-type GFP; the emission was also shifted to shorter wavelength. It may be noted that compared to the typical fluorophores formed in the natural and modified fluorescent proteins, the oxazole and thiazole fluorophores are completely stable and do not require activation by posttranslational modification to exhibit fluorescence.

Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro.

Plasmids containing 23S rRNA randomized at positions 2057-2063 and 2502-2507 were introduced into Escherichia coli, affording a library of clones which produced modified ribosomes in addition to the pre-existing wild-type ribosomes. These clones were screened with a derivative of puromycin, a natural product which acts as an analogue of the 3'-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puromycin derivative in this study contained the dipeptide p-methoxyphenylalanylglycine, implying the ability of the modified ribosomes in clones sensitive to this puromycin analogue to recognize dipeptides. Several clones inhibited by the puromycin derivative were used to make S-30 preparations, and some of these were shown to support the incorporation of dipeptides into proteins. The four incorporated species included two dipeptides (Gly-Phe (2) and Phe-Gly (3)), as well as a thiolated dipeptide analogue (4) and a fluorescent oxazole (5) having amine and carboxyl groups approximately the same distance apart as in a normal dipeptide. A protein containing both thiolated dipeptide 4 and a 7-methoxycoumarin fluorophore was found to undergo fluorescence quenching. Introduction of the oxazole fluorophore 5 into dihydrofolate reductase or green fluorescent protein resulted in quite strong enhancement of its fluorescence emission, and the basis for this enhancement was studied. The aggregate results demonstrate the feasibility of incorporating dipeptides as a single ribosomal event, and illustrate the lack of recognition of the central peptide bond in the dipeptide, potentially enabling the incorporation of a broad variety of structural analogues.

Protein Synthesis with Ribosomes Selected for the Incorporation of ß-Amino Acids.

In an earlier study, ß³-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different ß-amino acids into Escherichia coli dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate ß-amino acids into DHFR, in spite of the use of a single puromycin for the selection of the individual clones. In this study, we examine the extent to which the structure of the ß³-puromycin employed for ribosome selection influences the regio- and stereochemical preferences of the modified ribosomes during protein synthesis; the mechanistic probe was a single suppressor tRNA(CUA) activated with each of four methyl-ß-alanine isomers (1-4). The modified ribosomes were found to incorporate each of the four isomeric methyl-ß-alanines into DHFR but exhibited a preference for incorporation of 3(S)-methyl-ß-alanine (ß-mAla; 4), i.e., the isomer having the same regio- and stereochemistry as the O-methylated ß-tyrosine moiety of ß³-puromycin. Also conducted were a selection of clones that are responsive to ß²-puromycin and a demonstration of reversal of the regio- and stereochemical preferences of these clones during protein synthesis. These results were incorporated into a structural model of the modified regions of 23S rRNA, which included in silico prediction of a H-bonding network. Finally, it was demonstrated that incorporation of 3(S)-methyl-ß-alanine (ß-mAla; 4) into a short α-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties.

Incorporation of ß-amino acids into dihydrofolate reductase by ribosomes having modifications in the peptidyltransferase center.

Ribosomes containing modifications in three regions of 23S rRNA, all of which are in proximity to the ribosomal peptidyltransferase center (PTC), were utilized previously as a source of S-30 preparations for in vitro protein biosynthesis experiments. When utilized in the presence of mRNAs containing UAG codons at predetermined positions+ß-alanyl-tRNA(CUA), the modified ribosomes produced enhanced levels of full length proteins via UAG codon suppression. In the present study, these earlier results have been extended by the use of substituted ß-amino acids, and direct evidence for ß-amino acid incorporation is provided. Presently, five of the clones having modified ribosomes are used in experiments employing four substituted ß-amino acids, including α-methyl-ß-alanine, ß,ß-dimethyl-ß-alanine, ß-phenylalanine, and ß-(p-bromophenyl)alanine. The ß-amino acids were incorporated into three different positions (10, 18 and 49) of Escherichia coli dihydrofolate reductase (DHFR) and their efficiencies of suppression of the UAG codons were compared with those of ß-alanine and representative α-l-amino acids. The isolated proteins containing the modified ß-amino acids were subjected to proteolytic digestion, and the derived fragments were characterized by mass spectrometry, establishing that the ß-amino acids had been incorporated into DHFR, and that they were present exclusively in the anticipated peptide fragments. DHFR contains glutamic acid in position 17, and it has been shown previously that Glu-C endoproteinase can hydrolyze DHFR between amino acids residues 17 and 18. The incorporation of ß,ß-dimethyl-ß-alanine into position 18 of DHFR prevented this cleavage, providing further evidence for the position of incorporation of the ß-amino acid.

Synthesis and biological activities of topopyrones.

Structure-activity studies were employed to investigate the stabilization of DNA-topoisomerases I and II covalent binary complexes by topopyrone analogues. The synthesis of five new topopyrone derivatives and study of their ability to stabilize DNA-topoisomerase I and DNA-topoisomerase II covalent binary complexes are described. The biochemical assays suggest that the orientation of the fused 1,4-pyrone ring and halogen substituents contribute importantly to the overall potency of the topopyrones as topoisomerase poisons.