The C-terminal subdomain (IF2 C-2) contains the entire fMet-tRNA binding site of initiation factor IF2.
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-1) 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.
Spurio
R
R
Laboratory of Genetics, Department of Biology, University of Camerino, Camerino 62032, Italy.
Brandi
L
L
Caserta
E
E
Pon
C L
CL
Gualerzi
C O
CO
Misselwitz
R
R
Krafft
C
C
Welfle
K
K
Welfle
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D017931:DNA Primers; D019791:Guanidine; D006358:Hot Temperature; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D039881:Prokaryotic Initiation Factor-2; D011489:Protein Denaturation; D012358:RNA, Transfer, Met / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D012358:RNA, Transfer, Met; D011994:Recombinant Proteins; C024988:tRNA, formylmethionine-; D019791:Guanidine