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

Shijie Ye,Allison Ann Berger,Benjamin Matt,Beate Koksch,Oliver Reimann,Dominique Petzold

doi:10.3762/bjoc.6.40

Abstract

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.

Highlights

Over the past two decades, the interest in engineering proteins containing site-specific synthetic amino acids with novel functionalities has grown considerably
The utility of chemically aminoacylated suppressor transfer RNAs combined with cell-free translation systems in producing proteins that contain non-canonical amino acids was reported independently from each other by Schultz and Chamberlin [1,2]. Their methodology is based on the following observations: 1) the central intermediate molecule in protein translation, the aminoacyltRNA produced in the cell by specific tRNA synthetases can be semi-synthesized; 2) a nonsense codon TAG can replace an amino acid-encoding codon at a desired position and can be recognized by the corresponding mutated orthogonal suppressor tRNA during the translation (Figure 1)
We report here the chemical and enzymatic aminoacylation of the yeast phenylalanine suppressor tRNA with a series of fluoroalkylated amino acids for site-specific protein mutagenesis (Figure 2). (RS)-2-amino-2-methyl-3,3,3-trifluoropropanoic acid (α-(Tfm)Ala) [22], (S)-ethylglycine (Abu) and two of its fluorinated analogues, (S)-2-amino-4,4-difluorobutanoic acid (DfeGly) [23] and (S)-2-amino-4,4,4-trifluorobutanoic acid (TfeGly) [24], were synthesized in the appropriate protected activated form and used to chemically aminoacylate tRNAPheCUA by means of the hybrid dinucleotide pdCpA and enzymatic ligation

Summary

Introduction

Over the past two decades, the interest in engineering proteins containing site-specific synthetic amino acids with novel functionalities has grown considerably. 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. AminoacyltRNAs can be used to synthesize biologically relevant proteins which contain fluorinated amino acids at specific sites by means of a cell-free translation system.

Results

Conclusion