Changes in conformation and slow refolding kinetics in mutant iso-2-cytochrome c with replacement of a conserved proline residue.
Changes in conformation and slow refolding kinetics in mutant iso-2-cytochrome c with replacement of a conserved proline residue.
复制标题
通过替换保守的脯氨酸残基，突变体 iso-2-细胞色素 c 的构象变化和缓慢的重折叠动力学。
DOI：
10.1021/bi00388a026
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Nall,BT
中科院分区：
文献类型：
--
作者：
White,TB;Berget,PB;Nall,BT
修订稿于 1987 年 1 月 28 日收到 摘要：利用寡核苷酸定向诱变，我们产生了酵母 iso-2-细胞色素 c 的突变体，其中苏氨酸 (Thr-71) 取代了位于天然蛋白中两个短 a 螺旋片段之间的保守脯氨酸残基 (Pro-71)。光谱表明，在 pH 7.2 时，Thr-71 iso-2-细胞色素 c 折叠成非天然构象，可能与天然蛋白质的碱性形式有关。滴定至 pH 5.2 后，Thr-71 iso-2-细胞色素 c 恢复了正常蛋白质的许多光学特性。我们已经证明，71 位的脯氨酸残基对荧光检测的缓慢重折叠的动力学没有影响。然而，在 pH 5 和 pH 7.2 之间，吸光度检测到的缓慢折叠的幅度在突变蛋白中强烈依赖于 pH，但在正常蛋白中很大程度上独立于 pH。我们认为这是由于 Thr-71 iso-2-细胞色素 c 在 pH 7.2 下折叠成非天然构象，不需要在 pH 5-6 下折叠成更接近天然的状态时观察到的缓慢的、吸光度检测的构象变化。蛋白质的氨基酸序列以目前未知的方式决定其三维结构。为了了解特定氨基酸对结构形成过程的贡献，有必要确定单个氨基酸替换对蛋白质折叠特定动力学阶段的影响。类似地，特定氨基酸对天然蛋白质稳定性的贡献可以通过比较蛋白质的点突变体和野生型形式的平衡解折叠转变来确定。在本报告中，我们对脯氨酸711参与酵母酿酒酵母iso-2-细胞色素c（iso-2）2三级结构形成的部分分析提供了两种类型的数据。脯氨酸残基被认为在三级结构形成中发挥着重要作用。例如，酰亚胺键异构化被认为会导致蛋白质折叠的动力学阻断（Brandts 等，1975）。核糖核酸酶 A 的实验表明，未折叠群体的快折叠部分和慢折叠部分之间的平衡可以通过强酸催化，与尿素或盐酸胍浓度无关，并且活化焓为 21 kcal/mol（Schmid & Baldwin，1978；Schmid 等人，1984）。如果平衡由酰亚胺异构化控制，则这些性质是可以预期的。此外，人们可能期望动力学阶段和脯氨酸含量之间存在关系，事实上，11 个非同源蛋白质的重折叠动力学被证明与其脯氨酸含量有定性相关（Stellwagen，1979）。其他实验集中于比较不同的同源蛋白质
Revised Manuscript Received January 28, 1987 abstract: Using oligonucleotide-directed mutagenesis, we have produced a mutant form of iso-2-cytochrome c of yeast in which threonine (Thr-71) replaces a conserved proline residue (Pro-71) locatedbetween two short a-helical segments in the native protein. Optical spectroscopy indicates that, at pH 7.2, Thr-71 iso-2-cytochrome c folds to a nonnative conformation possibly related to the alkaline form of the native protein. On titration to pH 5.2, Thr-71 iso-2-cytochrome c regains many of the optical properties of the normal protein. We have shown that the proline residue at position 71 has no effect on the kinetics of fluorescence-detected slow refolding. However, between pH 5 and pH 7.2 the amplitude for absorbance-detectedslow folding is strongly pH dependent in the mutant protein but is largely independent of pH in the normal protein. We believe this to be due to the folding of Thr-71 iso-2-cytochrome c to a nonnative conformation at pH 7.2 that does notrequire the slow, absorbance-detected conformational changes observed in folding to the more native-like state at pH 5-6. e amino acidsequence of a protein determines its three-dimensional structure in a manner that is currently unknown. In order to understand the contribution of specific amino acids to the process of structure formation, it is necessary to identify the effects of single amino acid replacements on specific kinetic phases in protein folding. Similarly, the contributionof specific amino acids to the stability of the native protein can be de-termined by comparing equilibrium unfolding transitions for point mutant and wild-type forms of a protein. In this report, we present both types of data in a partial analysis of the involvement of proline-711 in the tertiary structure formation of iso-2-cytochrome c (iso-2) 2 from theyeast Saccharomyces cerevisiae.Proline residues are believed to play an important role in tertiary structure formation. Imide bond isomerization, for example, has been proposedto cause a kinetic block in protein folding (Brandts et al., 1975). Experiments with ribonuclease A have shown that equilibration between the fast and slow folding portions of an unfolded population can be catalyzed by strong acid, is independent of urea or guanidine hydrochloride concentrations, and has an activation enthalpy of 21 kcal/mol (Schmid & Baldwin, 1978; Schmid et al., 1984). These properties are those to be expected if the equilibration were governed by imide isomerization. In addition, one might expect a relationship between kinetic phases and proline content, and indeed, the refolding kinetics of 11 nonhomolo-gous proteins were shown to be qualtitatively related to their proline content (Stellwagen, 1979). Other experiments have concentrated on comparing homologous proteins that differ