Spurio R, Brandi L, Caserta E, Pon C L, Gualerzi C O, Misselwitz R, Krafft C, Welfle K, Welfle H
Laboratory of Genetics, Department of Biology, University of Camerino, Camerino 62032, Italy.
J Biol Chem. 2000 Jan 28;275(4):2447-54. doi: 10.1074/jbc.275.4.2447.
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.
先前的蛋白质解折叠研究表明，嗜热脂肪芽孢杆菌翻译起始因子IF2的24.5 kDa甲硫氨酸- tRNA结合结构域IF2 C可能由两个亚结构域组成。在本研究中，IF2 C的四个苯丙氨酸残基（第531、599、657和721位）被色氨酸取代，产生了四种在分子不同位置具有内在荧光标记的变体蛋白。通过比较圆二色性以及由盐酸胍浓度增加引起的色氨酸荧光变化，证明IF2 C确实由两个亚结构域组成：包含色氨酸-599的更稳定的N端（IF2 C-1）亚结构域和包含色氨酸-721的较不稳定的C端（IF2 C-2）亚结构域。发现仅由110个氨基酸（从Glu-632到Ala-741）组成的分离亚结构域IF2 C-2与天然IF2或IF2 C结构域以相同的特异性和亲和力结合甲硫氨酸- tRNA。从N端和C端修剪IF2 C-2表明，仍能够结合甲硫氨酸的最小片段由90个氨基酸组成。通过圆二色性、尿素、盐酸胍和温度诱导的解折叠以及差示扫描量热法对IF2 C-2进行了进一步表征。结果表明，IF2 C-2是一个主要包含β结构（25%反平行和8%平行β链）和转角（19%）的球状分子，其结构性质不受N端亚结构域IF2 C-1存在与否的严重影响。
