Perla-Kajan, J, Lin, X, Cooperman, BS, Goldman, E, Jakubowski, H, Knudsen, CR and Mandecki, W (2010) Properties of Escherichia coli EF-Tu mutants designed for fluorescence resonance energy transfer from tRNA molecules. Protein Eng. Des. Sel. 23:129-36
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.
Binding Sites; Electrophoretic Mobility Shift Assay; Escherichia coli/enzymology; Escherichia coli/genetics; Factor Xa/metabolism; Fluorescence Resonance Energy Transfer; Guanosine Diphosphate/metabolism; Hydrolysis; Models, Molecular; Mutagenesis, Site-Directed; Mutant Proteins/chemistry; Mutant Proteins/genetics; Mutant Proteins/isolation & purification; Mutant Proteins/metabolism; Mutation; Nucleic Acid Conformation; Peptide Elongation Factor Tu/chemistry; Peptide Elongation Factor Tu/genetics; Peptide Elongation Factor Tu/isolation & purification; Peptide Elongation Factor Tu/metabolism; Peptides/metabolism; Protein Conformation; Protein Engineering; RNA, Transfer/chemistry; RNA, Transfer/metabolism; Sequence Analysis, DNA; Staining and Labeling
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