Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark.
Biochem Biophys Res Commun. 2011 Dec 9;416(1-2):140-5. doi: 10.1016/j.bbrc.2011.11.012. Epub 2011 Nov 11.
Translation initiation factor 5B (IF5B) is required for initiation of protein synthesis. The solution structure of archaeal IF5B (aIF5B) was analysed by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) and was indicated to be in both monomeric and dimeric form. Sedimentation equilibrium (SE) analytical ultracentrifugation (AUC) of aIF5B indicated that aIF5B forms irreversible dimers in solution but only to a maximum of 5.0-6.8% dimer. Sedimentation velocity (SV) AUC at higher speed also indicated the presence of two species, and the sedimentation coefficients s(20,w)(0) were determined to be 3.64 and 5.51±0.29 S for monomer and dimer, respectively. The atomic resolution (crystallographic) structure of aIF5B (Roll-Mecak et al. [6]) was used to model monomer and dimer, and theoretical sedimentation coefficients for these models were computed (3.89 and 5.63 S, respectively) in good agreement with the sedimentation coefficients obtained from SV analysis. Thus, the structure of aIF5B in solution must be very similar to the atomic resolution structure of aIF5B. SAXS data were acquired in the same buffer with the addition of 2% glycerol to inhibit dimerization, and the resultant monomeric aIF5B in solution did indeed adopt a structure very similar to the one reported earlier for the protein in crystalline form. The p(r) function indicated an elongated conformation supported by a radius of gyration of 37.5±0.2 Å and a maximum dimension of ~130 Å. The effects of glycerol on the formation of dimers are discussed. This new model of aIF5B in solution shows that there are universal structural differences between aIF5B and the homologous protein IF2 from Escherichia coli.
翻译起始因子 5B（IF5B）是蛋白质合成起始所必需的。通过小角 X 射线散射（SAXS）和动态光散射（DLS）分析了古菌 IF5B（aIF5B）的溶液结构，表明其处于单体和二聚体两种形式。aIF5B 的沉降平衡（SE）分析超速离心（AUC）表明，aIF5B 在溶液中形成不可逆的二聚体，但最多只能形成 5.0-6.8%的二聚体。在更高速度的沉降速度（SV）AUC 也表明存在两种物质，沉降系数 s（20，w）（0）分别确定为单体和二聚体的 3.64 和 5.51±0.29 S。aIF5B 的原子分辨率（晶体学）结构（Roll-Mecak 等人[6]）用于模拟单体和二聚体，并且计算了这些模型的理论沉降系数（分别为 3.89 和 5.63 S），与 SV 分析获得的沉降系数非常吻合。因此，溶液中 aIF5B 的结构必须与 aIF5B 的原子分辨率结构非常相似。在添加 2%甘油以抑制二聚化的相同缓冲液中获得 SAXS 数据，并且溶液中确实形成了单体 aIF5B，其结构与以前报道的蛋白质晶体形式非常相似。p(r) 函数表明，一种由回转半径为 37.5±0.2 Å 和最大尺寸约 130 Å 的旋转椭球体支持的伸长构象。讨论了甘油对二聚体形成的影响。该溶液中 aIF5B 的新模型表明，aIF5B 与同源蛋白大肠杆菌 IF2 之间存在普遍的结构差异。
