Stability of a 24-meric homopolymer:: Comparative studies of assembly-defective mutants of Rhodobacter capsulatus bacterioferritin and the native protein
Stability of a 24-meric homopolymer:: Comparative studies of assembly-defective mutants of Rhodobacter capsulatus bacterioferritin and the native protein
复制标题
DOI：
10.1110/ps.0301903
复制
发表时间：
2003-08-01
期刊：
影响因子：
8
通讯作者：
Moore, GR
中科院分区：
文献类型：
--
作者：
Kilic, MA;Spiro, S;Moore, GR
使用非变性 PAGE、CD 和荧光光谱研究了荚膜红杆菌细菌铁蛋白（一种 24 聚体均聚物）在 pH 和温度变化以及尿素和盐酸胍浓度增加时变性的稳定性。在温度和尿素的作用下，野生型蛋白质在平衡实验中变性，没有可辨别的中间体，但使用盐酸胍 (Gnd.HCl) 时，在相对较低的 Gnd.HCl 浓度下，一种或多种中间体物质是明显的。如果不存在高比例的 24 聚体，则在任何 pH 下都无法获得解离的亚基单体或小于 24 聚体的聚集体，其中含有天然蛋白质的高 α 螺旋含量特征，并且与其他变性实验和通过定点诱变构建稳定的亚基二聚体结合起来，该观察结果表明细菌铁蛋白单体的折叠可以与其结合成二聚体。 Glu 128 和 Glu。在一系列突变体中，135被丙氨酸和精氨酸取代，以确定它们在稳定24聚寡聚物中的作用。 Glu128Ala、Glu135Ala 和 Glu135Arg 变体保留了 24 聚体结构，但 Glu128Ala/Glu135Ala 和 Glu128Arg/Glu135Arg 变体是稳定的亚基二聚体。 Glu135Arg、Glu128AIa/Glu135Ala 和 Glu128Arg/Glu135Arg 变体的 CD 谱表明它们保留了野生型蛋白质的高 α 螺旋含量。 24 聚体 Glu135Arg 变体比野生型蛋白（59°C 的 T-m、[Urea](50%) 和 [Gnd.HCl](50%)，4.9 M 和 3.2 M 与 73°C 相比，分别与 8 M 和 4.3 M 相似）稳定性较差，二聚体 Glu128Arg/Glu135Arg 变体的稳定性仍然较差（T-m，[尿素]（50%）和[Gnd.HCl]（50%），43℃，分别类似于3.2 M和1.8 M）。稳定性差异大致是相加的，表明天然寡聚物中的 Glu 128 和 Glu 135 分别与 Arg 61 和相邻亚基的氨基末端胺形成的盐桥对亚基组装的稳定性有同等贡献。变体蛋白的加和性和组装状态表明，涉及 Glu 128 和 Glu 135 的相互作用对于稳定 24 聚体相对于亚基二聚体有显着贡献。
The stability of Rhodobacter capsulatus bacterioferritin, a 24-meric homopolymer, toward denaturation on variation in pH and temperature, and increasing concentrations of urea and guanidine.HCl was investigated with native PAGE, and CD and fluorescence spectroscopies. With temperature and urea, the wild-type protein denatured without discernible intermediates in the equilibrium experiments, but with guanidine.HCl (Gnd.HCl) one or more intermediate species were apparent at relatively low Gnd.HCl concentrations. Dissociated subunit monomers, or aggregates smaller than 24-mers containing the high alpha-helical content characteristic of the native protein were not obtained at any pH without a high proportion of the 24-mer being present, and taken together with the other denaturation experiments and the construction of stable subunit dimers by site-directed mutagenesis, this observation indicates that folding of the bacterioferritin monomer could be coupled to its association into a dimer. Glu 128 and Glu. 135 were replaced by alanine and arginine in a series of mutants to determine their role in stabilizing the 24-meric oligomer. The Glu128Ala, Glu135Ala and Glu135Arg variants retained a 24-meric structure, but the Glu128Ala/Glu135Ala and Glu128Arg/Glu135Arg variants were stable subunit dimers. CD spectra of the Glu135Arg, Glu128AIa/Glu135Ala, and Glu128Arg/Glu135Arg variants showed that they retained the high alpha-helical content of the wild-type protein. The 24-meric Glu135Arg variant was less stable than the wild-type protein (T-m, [Urea](50%) and [Gnd.HCl](50%), of 59degreesC, 4.9 M and 3.2 M compared with 73degreesC, similar to8 M and 4.3 M, respectively), and the dimeric Glu128Arg/Glu135Arg variant was less stable still (T-m, [Urea](50%) and [Gnd.HCl](50%). of 43degreesC, similar to3.2 M and 1.8 M, respectively). The differences in stability are roughly additive, indicating that the salt-bridges formed by Glu 128 and Glu 135 in the native oligomer, with Arg 61 and the amino-terminal amine of neighboring subunits, respectively, contribute equally to the stability of the subunit assembly. The additivity and assembly states of the variant proteins suggest that the interactions involving Glu 128 and Glu 135 contribute significantly to stabilizing the 24-mer relative to the subunit dimer.