Replacement of a conserved proline eliminates the absorbance-detected slow folding phase of iso-2-cytochrome c.
Replacement of a conserved proline eliminates the absorbance-detected slow folding phase of iso-2-cytochrome c.
复制标题
保守脯氨酸的替换消除了吸光度检测到的 iso-2-细胞色素 c 的慢折叠阶段。
DOI：
10.1021/bi00423a009
复制
发表时间：
1988
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：
Wood,LC;White,TB;Ramdas,L;Nall,BT
德克萨斯大学健康科学中心生物化学系，圣安东尼奥，德克萨斯州78284；德克萨斯大学医学院生物化学和分子生物学系，休斯顿，德克萨斯州77225摘要：作为脯氨酸异构化模型的测试，我们使用寡核苷酸位点定向诱变技术构建了一种异-2-细胞色素c的突变体，其中脯氨酸-76被甘氨酸取代[Wood， L. c ., Muthukrishnan， K., White， T. B., Ramdas， L., &Nall， B. T.(1988) Biochemistry（本期的前一篇论文）]。对于氧化形式的Gly-76 iso-2，通过盐酸胍诱导展开的稳定性估计表明，在中性pH和20℃的标准条件下，突变使蛋白质不稳定1.2 kcal/mol（正常Pro-76iso-2的AGU= 3.8 kcal/mol，而Gly-76 iso-2的AGU= 2.6 kcal/mol）。折叠/展开的动力学已通过荧光变化监测整个过渡区使用停止流动混合。荧光检测的快速和慢速重折叠速率不变，而与正常的iso-2相比，突变蛋白的快速展开速率增加了3倍。荧光检测的突变蛋白展开在1-s时间范围内观察到一个新的动力学相。新阶段的存在与初始条件下折叠构象改变的物种的存在相关，表明该阶段与该物种的展开有关。通过在pH 5.5和8 （0.3 M盐酸胍，20℃）之间手动混合，监测荧光检测和吸光度检测的慢折叠相作为最终pH的函数。荧光检测慢折叠的振幅和速率与pH值无关，与正常iso-2的荧光检测慢折叠难以区分。在Gly-76 iso-2的折叠中没有通常的吸收检测到的慢折叠相，这表明正常的iso-2的吸收检测到的慢折叠种是由未折叠蛋白中的脯氨酸-76亚胺键的异构化产生的。通过吸光度变化检测到的一个新的慢动力学相被证明是折叠的本地样物种和突变结构改变之间的构象变化。我们提出，突变蛋白成功折叠成改变的构象，一般来说，在通过构象变化转化为改变的三维结构之前，可能遵循到原生状态的途径。
Department of Biochemistry, The University of Texas Health Science Center, SanAntonio, Texas 78284, and Department of Biochemistry and Molecular Biology, The University of Texas Medical School, Houston, Texas 77225 Received April 6, 1988; Revised Manuscript Received July 13, 1988 abstract: As a test of the proline isomerization model, we have used oligonucleotide site-directed mutagenesis to construct a mutant form of iso-2-cytochrome c in which proline-76 is replaced by glycine [Wood, L. C., Muthukrishnan, K., White, T. B., Ramdas, L., &Nall, B. T.(1988) Biochemistry (preceding paper in this issue)]. For the oxidized form of Gly-76 iso-2, an estimate of stability by guanidine hydrochloride induced unfolding indicates that the mutation destabilizes the protein by 1.2 kcal/mol under standard conditions of neutral pH and 20 C (AGU= 3.8 kcal/mol fornormal Pro-76iso-2 versus 2.6 kcal/mol for Gly-76 iso-2). The kinetics of folding/unfolding have been monitored by fluorescence changes throughout the transition region using stopped-flow mixing. Therates for fastand slow fluorescence-detected refolding are unchanged, while fast unfolding is increased in rate 3-fold in the mutant protein compared to normal iso-2. A new kinetic phase in the 1-s time range is observed in fluorescence-detected unfoldingof the mutant protein. The presence of the new phase is correlated with the presence of species with an altered folded conformation in the initial conditions, suggesting assignment of the phase to unfolding of this species. The fluorescence-detected and absorbance-detected slow folding phases have been monitored as a function of final pH by manualmixing between pH 5.5 and 8 (0.3 M guanidine hydrochloride, 20 C). Both the amplitudes and rates for fluorescence-detected slow folding are independent of pH and are indistinguishable from fluorescence-detected slow foldingof normal iso-2. The usual absorbance-detected slow folding phase is absent in folding of Gly-76 iso-2, suggesting that theabsorbance-detected slow folding species for normal iso-2 are generated by isomerization of the proline-76 imide bond in the unfolded protein. A new slow kinetic phase detected by absorbance changes is shown to be a conformational change between folded nativelike species and an altered mutant structure. We propose that successful folding of mutant proteins to altered conformations may, in general, follow the pathway to the nativelike state prior to conversion to altered three-dimensional structures via conformational changes.