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

Abstract

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 preexisting 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.

Synthesis and Evaluation of a Library of Fluorescent Dipeptidomimetic Analogues as Substrates for Modified Bacterial Ribosomes

“…It may be noted that oxazole 1a has previously been incorporated into DHFR in suppression yields of up to 15%. 12 …”

“…12,13 The incorporations have been verified by mass spectrometric analysis of tryptic digests of the modified proteins. The strongly fluorescent nature of the derived proteins prompted the preparation and study of a library of substituted oxazoles and thiazoles to permit a better understanding of the nature and scope of our initial observations.…”

“…Thus, introducing 1a at position 39 of GFP produced a protein ~2-fold brighter than native GFP, while introducing this fluorophore at position 66 resulted in a protein ~4-fold brighter than wild-type GFP. 12 This change was associated with a change in the absorption extinction coefficient of 1b . Embedding oxazole 1a in GFP at position 66 enhanced its brightness ~25–30-fold.…”

“…The syntheses of the pdCpA derivatives of the dipeptidomimetic analogues 1a and 8a and acceptor Acd have been reported previously. 12–14 The experimental procedures and compound characterizations are provided in the Supporting Information.…”

“…Accordingly, the modified ribosomes were capable of incorporating a thiodipeptide, as well as fluorescent oxazole ( 1a ) and thiazole ( 8a ) heterocycles (Figure 1) substituted with NH 2 and COOH groups at distances that approximated those between such functional groups in a dipeptide. 12,13 …”