Properties of Escherichia coli EF-Tu mutants designed for fluorescence resonance energy transfer from tRNA molecules

Joanna Perla-Kajan, Xin Lin, Barry S. Cooperman, Emanuel Goldman, Hieronim Jakubowski, Charlotte R. Knudsen, Wlodek Mandecki

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

Here we describe the design, preparation and characterization of 10 EF-Tu mutants of potential utility for the study of Escherichia coli elongation factor Tu (EF-Tu) interaction with tRNA by a fluorescence resonance energy transfer assay. Each mutant contains a single cysteine residue at positions in EF-Tu that are proximal to tRNA sites within the aminoacyl-tRNA·EF-Tu·GTP ternary complex that have previously been labeled with fluorophores. These positions fall in the 323-326 and 344-348 regions of EF-Tu, and at the C terminus. The EF-Tus were isolated as N-terminal fusions to glutathione S-transferase (GST), which was cleaved to yield intact EF-Tus. The mutant EF-Tus were tested for binding to GDP, binding to tRNA in gel retardation and protection assays, and activity in poly-U translation in vitro. The results indicate that at least three EF-Tu mutants, K324C, G325C and E348C, are suitable for further studies. Remarkably, GST fusions that were not cleaved were also active in the various assays, despite the N-terminal fusion.

Original languageEnglish (US)
Pages (from-to)129-136
Number of pages8
JournalProtein Engineering, Design and Selection
Volume23
Issue number3
DOIs
StatePublished - Mar 2010

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

Keywords

  • Elongation factor Tu
  • FRET
  • Site-directed mutagenesis
  • Structure-function
  • Transfer RNA

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