Experimental Evolution of a Green Fluorescent Protein Composed of 19 Unique Amino Acids without Tryptophan

Akio Kawahara-Kobayashi, Mitsuhiro Hitotsuyanagi, Kazuaki Amikura, Daisuke Kiga

Research output: Contribution to journalArticle

Abstract

At some stage of evolution, genes of organisms may have encoded proteins that were synthesized using fewer than 20 unique amino acids. Similar to evolution of the natural 19-amino-acid proteins GroEL/ES, proteins composed of 19 unique amino acids would have been able to evolve by accumulating beneficial mutations within the 19-amino-acid repertoire encoded in an ancestral genetic code. Because Trp is thought to be the last amino acid included in the canonical 20-amino-acid repertoire, this late stage of protein evolution could be mimicked by experimental evolution of 19-amino-acid proteins without tryptophan (Trp). To further understand the evolution of proteins, we tried to mimic the evolution of a 19-amino-acid protein involving the accumulation of beneficial mutations using directed evolution by random mutagenesis on the whole targeted gene sequence. We created active 19-amino-acid green fluorescent proteins (GFPs) without Trp from a poorly fluorescent 19-amino-acid mutant, S1-W57F, by using directed evolution with two rounds of mutagenesis and selection. The N105I and S205T mutations showed beneficial effects on the S1-W57F mutant. When these two mutations were combined on S1-W57F, we observed an additive effect on the fluorescence intensity. In contrast, these mutations showed no clear improvement individually or in combination on GFPS1, which is the parental GFP mutant composed of 20 amino acids. Our results provide an additional example for the experimental evolution of 19-amino-acid proteins without Trp, and would help understand the mechanisms underlying the evolution of 19-amino-acid proteins. (236 words).

Original languageEnglish
Pages (from-to)75-86
Number of pages12
JournalOrigins of Life and Evolution of the Biosphere
Volume44
Issue number2
DOIs
Publication statusPublished - 2014 Dec 11
Externally publishedYes

Fingerprint

tryptophan
Green Fluorescent Proteins
green fluorescent protein
Tryptophan
amino acids
amino acid
proteins
Amino Acids
protein
mutations
mutation
Proteins
mutagenesis
Mutation
Mutagenesis
genes
mutants
genetic code
Chaperonin 60
Genetic Code

Keywords

  • 19 amino acids
  • Directed evolution
  • Genetic code
  • Green fluorescent protein
  • Tryptophan

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Space and Planetary Science
  • Medicine(all)

Cite this

Experimental Evolution of a Green Fluorescent Protein Composed of 19 Unique Amino Acids without Tryptophan. / Kawahara-Kobayashi, Akio; Hitotsuyanagi, Mitsuhiro; Amikura, Kazuaki; Kiga, Daisuke.

In: Origins of Life and Evolution of the Biosphere, Vol. 44, No. 2, 11.12.2014, p. 75-86.

Research output: Contribution to journalArticle

Kawahara-Kobayashi, Akio ; Hitotsuyanagi, Mitsuhiro ; Amikura, Kazuaki ; Kiga, Daisuke. / Experimental Evolution of a Green Fluorescent Protein Composed of 19 Unique Amino Acids without Tryptophan. In: Origins of Life and Evolution of the Biosphere. 2014 ; Vol. 44, No. 2. pp. 75-86.
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