Kilic Mehmet A, Spiro Stephen, Moore Geoffrey R
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
Protein Sci. 2003 Aug;12(8):1663-74. doi: 10.1110/ps.0301903.
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, Glu128Ala/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 and Gnd.HCl of 59 degrees C, 4.9 M and 3.2 M compared with 73 degrees C, approximately 8 M and 4.3 M, respectively), and the dimeric Glu128Arg/Glu135Arg variant was less stable still (T(m), Urea and Gnd.HCl of 43 degrees C, approximately 3.2 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.
球形红杆菌细菌铁蛋白是一种由24个亚基组成的同聚物，利用天然聚丙烯酰胺凝胶电泳、圆二色光谱和荧光光谱研究了其在pH值、温度变化以及尿素和盐酸胍浓度增加时的变性稳定性。对于温度和尿素，在平衡实验中野生型蛋白变性时没有可识别的中间体，但对于盐酸胍（Gnd.HCl），在相对较低的Gnd.HCl浓度下会出现一个或多个中间物种。在任何pH值下，若不存在高比例的24聚体，则无法获得解离的亚基单体或小于24聚体的聚集体，而这些聚集体含有天然蛋白特有的高α-螺旋含量。结合其他变性实验以及通过定点诱变构建稳定的亚基二聚体，这一观察结果表明细菌铁蛋白单体的折叠可能与其缔合成二聚体有关。在一系列突变体中，将Glu 128和Glu 135分别替换为丙氨酸和精氨酸，以确定它们在稳定24聚体寡聚物中的作用。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、Glu128Ala/Glu135Ala和Glu128Arg/Glu135Arg变体的圆二色光谱表明它们保留了野生型蛋白的高α-螺旋含量。24聚体的Glu135Arg变体比野生型蛋白稳定性低（其熔点温度、50%尿素浓度和50%盐酸胍浓度分别为59℃、4.9 M和3.2 M，而野生型分别为73℃、约8 M和4.3 M），二聚体的Glu128Arg/Glu135Arg变体稳定性更低（其熔点温度、50%尿素浓度和50%盐酸胍浓度分别为43℃、约3.2 M和1.8 M）。稳定性差异大致呈加和性，表明天然寡聚物中由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贡献相同。变体蛋白的加和性和组装状态表明，相对于亚基二聚体，涉及Glu 128和Glu 135的相互作用对稳定24聚体有显著贡献。
