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The C-terminal subdomain (IF2 C-2) contains the entire fMet-tRNA binding site of initiation factor IF2

Item Type:Article
Title:The C-terminal subdomain (IF2 C-2) contains the entire fMet-tRNA binding site of initiation factor IF2
Creators Name:Spurio, R., Brandi, L., Caserta, E., Pon, C.L., Gualerzi, C.O., Misselwitz, R., Krafft, C., Welfle, K. and Welfle, H.
Abstract:Previous protein unfolding studies had suggested that IF2 C, the 24.5- kDa fMet-tRNA binding domain of Bacillus stearothermophilus translation initiation factor IF2, may consist of two subdomains. In the present work, the four Phe residues of IF2 C (positions 531, 599, 657, and 721) were replaced with Trp, yielding four variant proteins having intrinsic fluorescence markers in different positions of the molecule. Comparison of the circular dichroism and Trp fluorescence changes induced by increasing concentrations of guanidine hydrochloride demonstrated that IF2 C indeed consists of two subdomains: the more stable N-terminal (IF2 C-l) subdomain containing Trp-599, and the less stable C-terminal (IF2 C-2) subdomain containing Trp-721. Isolated subdomain IF2 C-2, which consists of just 110 amino acids (from Glu-632 to Ala-741), was found to bind fMet-tRNA with the same specificity and affinity as native IF2 or IF2 C-domain. Trimming IF2 C-2 from both N and C termini demonstrated that the minimal fragment still capable of fMet-binding consists of 90 amino acids. IF2 C-2 was further characterized by circular dichroism; by urea-, guanidine hydrochloride-, and temperature-induced unfolding; and by differential scanning calorimetry. The results indicate that IF2 C-2 is a globular molecule containing predominantly {beta} structures (25% antiparallel and 8% parallel {beta} strands) and turns (19%) whose structural properties are not grossly affected by the presence or absence of the N-terminal subdomain IF2 C-1.
Keywords:Amino Acid Sequence, Base Sequence, Binding Sites, Differential Scanning Calorimetry, DNA Primers, Fluorescence Spectrometry, Guanidine, Heat, Met Transfer RNA, Peptide Initiation Factors, Prokaryotic Initiation Factor-2, Protein Denaturation, Recombinant Proteins
Source:Journal of Biological Chemistry
Publisher:American Society for Biochemistry and Molecular Biology
Page Range:2447-2454
Date:28 January 2000
Official Publication:http://www.jbc.org/cgi/content/abstract/275/4/2447
PubMed:View item in PubMed

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