Use of a spectrophotometric assay to evaluate amino acid activation and tRNA charging

Abstract

Conventional assays that investigate amino acid activation and tRNA‐charging rely on using radiolabeled amino acids (14C) and tRNAs (32P) to detect the presence of activated amino acid adenylate(s) and acylated tRNAs. There are also several assays that monitor activation and acylation products by using (32P) radiolabeled ATP and inorganic pyrophosphate. The radioisotope based assays offer unmatched sensitivity. However, the experiments require the use of hazardous reagents and often laborious protocols. More recently a colorimetric based assay has been developed to detect the level of inorganic free phosphate in aqueous solution. The assay is based on the complexation of malachite green molybdate and inorganic phosphate – the complex absorbs at 620 nm. The assay, referred to as the malachite green assay, has been modified to measure amino acid activation and tRNA charging. Our short‐term goal is to use the malachite green assay to measure the ability of aminoacyl‐tRNA syntheses (aaRSs) to activate and charge analog amino acids (AAs). Analog or “unnatural” AAs are non‐proteinogenic residues beyond the set of natural 20 L‐AAs. Bacteria and fungi use large multi‐subunit enzyme complexes, nonribosomal peptide synthetases (NRPs), to generate peptides composed of analog AAs. The resulting analog peptides typically possess enhanced bioactivity. Analog peptide bioactivity is generally linked with enhanced: i)membrane permeability, ii)protease stability and iii)chemical complexity. These advantages make analog peptide ideal for biological therapeutics. Our long‐term goal is to use the cell free expression system – the PURE system ( Protein synthesis Using Recombinant Elements) to synthesize analog peptides. The resulting peptides will be directed against protein‐protein interactions known to exacerbate disease. Our initial studies focus on using the malachite green assay to screen analog amino acids for activation and tRNA charging using purified aaRS and tRNA (total tRNA and single isoacceptors). Analogs identified via screening will be assembled into a library that will be used to generate analog peptides.Support or Funding InformationUniversity of San Diego