Sha M, Wang Y, Xiang T, van Heerden A, Browning K S, Goss D J
Department of Chemistry, Hunter College, City University of New York, New York 10021-5024, USA.
J Biol Chem. 1995 Dec 15;270(50):29904-9. doi: 10.1074/jbc.270.50.29904.
The binding of p28, p86, and native wheat germ eIF-(iso)4F with m7GTP and oligonucleotides was measured and compared. The purified subunits (p28, 28 kDa and p86, 86 kDa) of wheat germ protein synthesis initiation factor eIF-(iso)4F have been obtained from Escherichia coli expression of the cloned DNA (van Heerden, A., and Browning, K. S. (1994) J. Biol. Chem. 269, 17454-17457). The binding of the 5'-terminal cap analogue m7GTP to the small subunit (p28) of eIF-(iso)4F as a function of pH, temperature, and ionic strength is described. The mode of binding of p28 to cap analogues is very similar to the intact protein. Assuming that all tryptophan residues contribute to p28 and eIF-(iso)4F fluorescence, iodide quenching shows that all 9 tryptophan residues in p28 are solvent-accessible, while only 6 out of 16 tryptophan residues are solvent-accessible on the intact eIF-(iso)4F. The fluorescence stopped-flow studies of eIF-(iso)4F and p28 with cap show a concentration-independent conformational change. The rate of this conformational change was approximately 10-fold faster for the isolated p28 compared with the native eIF-(iso)4F. From these studies it appears that cap recognition resides in the p28 subunit. However, p86 enhances the interaction with capped oligonucleotides and probably is involved in protein-protein interactions as well. Both subunits are required for helicase activity.
测定并比较了p28、p86和天然小麦胚eIF-(iso)4F与m7GTP及寡核苷酸的结合情况。从小麦胚蛋白质合成起始因子eIF-(iso)4F纯化得到的亚基（p28，28 kDa；p86，86 kDa）是通过克隆DNA在大肠杆菌中的表达获得的（范·海尔登，A.，和布朗宁，K. S.（1994年）《生物化学杂志》269，17454 - 17457）。描述了5'-末端帽类似物m7GTP与eIF-(iso)4F小亚基（p28）的结合随pH、温度和离子强度的变化情况。p28与帽类似物的结合模式与完整蛋白非常相似。假设所有色氨酸残基都对p28和eIF-(iso)4F的荧光有贡献，碘化物淬灭表明p28中的所有9个色氨酸残基都可与溶剂接触，而完整的eIF-(iso)4F中16个色氨酸残基中只有6个可与溶剂接触。eIF-(iso)4F和p28与帽的荧光停流研究显示出浓度无关的构象变化。与天然eIF-(iso)4F相比，分离的p28这种构象变化的速率快约10倍。从这些研究看来，帽识别存在于p28亚基中。然而，p86增强了与带帽寡核苷酸的相互作用，并且可能也参与蛋白质 - 蛋白质相互作用。两个亚基都是解旋酶活性所必需的。
