Abstract
The 18-kDa Domain I from the N-terminal region of translation initiation factor IF2 from Escherichia coli was expressed, purified, and structurally characterized using multidimensional NMR methods. Residues 2-50 were found to form a compact subdomain containing three short beta-strands and three alpha-helices, folded to form a betaalphaalphabetabetaalpha motif with the three helices packed on the same side of a small twisted beta-sheet. The hydrophobic amino acids in the core of the subdomain are conserved in a wide range of species, indicating that a similarly structured motif is present at the N terminus of IF2 in many of the bacteria. External to the compact 50-amino acid subdomain, residues 51-97 are less conserved and do not appear to form a regular structure, whereas residues 98-157 form a helix containing a repetitive sequence of mostly hydrophilic amino acids. Nitrogen-15 relaxation rate measurements provide evidence that the first 50 residues form a well ordered subdomain, whereas other regions of Domain I are significantly more mobile. The compact subdomain at the N terminus of IF2 shows structural homology to the tRNA anticodon stem contact fold domains of the methionyl-tRNA and glutaminyl-tRNA synthetases, and a similar fold is also found in the B5 domain of the phenylalanine-tRNA synthetase. The results of the present work will provide guidance for the design of future experiments directed toward understanding the functional roles of this widely conserved structural domain within IF2.
Highlights
The 18-kDa Domain I from the N-terminal region of translation initiation factor IF2 from Escherichia coli was expressed, purified, and structurally characterized using multidimensional nuclear magnetic resonance (NMR) methods
circular dichroism (CD) spectra recorded at 30 °C or below look essentially the same, whereas spectra recorded at higher temperatures differ significantly, presumably due to unfolding of the protein (Fig. 2B)
The protein can be reversibly denatured by heating; a sample heated to 70 °C and cooled to 20 °C has a CD spectrum that is the same as that recorded before heating of the sample
Summary
Protein Cloning, Expression, and Purification—The fragment of the infB gene encoding the first domain of IF2–1 was amplified by PCR using E. coli K12 as template and primers that included unique restriction sites for XbaI and NdeI for insertion into the pET-15b expression vector (Novagen). Additional NOE cross-peaks were identified in the three-dimensional 15N- and 13C-edited NOE spectra (60-ms mixing time) and assigned to distance restraints as strong (Ͻ5.0 Å), medium (Ͻ5.5 Å), weak (Ͻ6.3 Å), and very weak (Ͻ6.9 Å). A very conservative distance restraint of Ͻ7.9 Å was used for NOE cross-peaks identified in spectra obtained with a relatively long mixing time (160 ms), where the effects of spin diffusion are most likely to be present. The repetitive amino acid sequence of residues 98 –157 prevented complete resonance assignments due to overlap in the spectra; those NOEs that are clearly resolved, as well as the CSI values of the overlapping peaks, suggest a helical structure for these 60 residues. Rotational correlation times and order parameters were calculated using Modelfree 4.0 [27] as previously described [28]
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