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

Rumit Maini, Larisa Dedkova, Rakesh Paul, Manikandadas M. Madathil, Sandipan Roy Chowdhury, Shengxi Chen, Sidney Hecht

Research output: Contribution to journalArticle

22 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)11206-11209
Number of pages4
JournalJournal of the American Chemical Society
Volume137
Issue number35
DOIs
StatePublished - Sep 9 2015

Fingerprint

Dipeptides
Ribosomes
Proteins
Puromycin
Clone Cells
Oxazoles
Fluorophores
Derivatives
glycylphenylalanine
phenylalanylglycine
Fluorescence
In Vitro Techniques
Tetrahydrofolate Dehydrogenase
Peptides
Polypeptides
Transfer RNA
Green Fluorescent Proteins
Biological Products
Escherichia coli
Amines

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro. / Maini, Rumit; Dedkova, Larisa; Paul, Rakesh; Madathil, Manikandadas M.; Chowdhury, Sandipan Roy; Chen, Shengxi; Hecht, Sidney.

In: Journal of the American Chemical Society, Vol. 137, No. 35, 09.09.2015, p. 11206-11209.

Research output: Contribution to journalArticle

Maini, Rumit ; Dedkova, Larisa ; Paul, Rakesh ; Madathil, Manikandadas M. ; Chowdhury, Sandipan Roy ; Chen, Shengxi ; Hecht, Sidney. / Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro. In: Journal of the American Chemical Society. 2015 ; Vol. 137, No. 35. pp. 11206-11209.
@article{91557f8e3db84cf8ab3212bb52cd9391,
title = "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 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.",
author = "Rumit Maini and Larisa Dedkova and Rakesh Paul and Madathil, {Manikandadas M.} and Chowdhury, {Sandipan Roy} and Shengxi Chen and Sidney Hecht",
year = "2015",
month = "9",
day = "9",
doi = "10.1021/jacs.5b03135",
language = "English (US)",
volume = "137",
pages = "11206--11209",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "35",

}

TY - JOUR

T1 - Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro

AU - Maini, Rumit

AU - Dedkova, Larisa

AU - Paul, Rakesh

AU - Madathil, Manikandadas M.

AU - Chowdhury, Sandipan Roy

AU - Chen, Shengxi

AU - Hecht, Sidney

PY - 2015/9/9

Y1 - 2015/9/9

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84941242156&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84941242156&partnerID=8YFLogxK

U2 - 10.1021/jacs.5b03135

DO - 10.1021/jacs.5b03135

M3 - Article

VL - 137

SP - 11206

EP - 11209

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 35

ER -