Characterization of a partially unfolded structure of cytochrome c induced by sodium dodecyl sulphate and the kinetics of its refolding
Characterization of a partially unfolded structure of cytochrome c induced by sodium dodecyl sulphate and the kinetics of its refolding
复制标题
DOI：
10.1046/j.1432-1327.1998.2540662.x
复制
发表时间：
1998-06-15
期刊：
影响因子：
--
通讯作者：
Mitra, S
中科院分区：
文献类型：
--
作者：
Das, TK;Mazumdar, S;Mitra, S
从血红素吸收、色氨酸荧光、圆二色性、共振拉曼散射、停止流动和时间分辨共振能量转移等方面研究了表面活性剂十二烷基硫酸钠诱导铁色素c展开的机理，全面了解了整个过程。在本研究的浓度范围（20-50 μ M）内，SDS/细胞色素c几乎以特定的分子比例展开。然而，当细胞色素c浓度较低时，在大约0.6 mM SDS处似乎有一个点开始展开。展开动力学显示，只有一次转变，速率常数为33 s(-1)（在298 K， [SDS] = 8.7 mM），活化能势垒约为16 kJ/mol，表明其他相关步骤，如果有的话，太快了，无法显著填充。与展开跃迁相关的自由能变化（G度）估计约为16.8 kJ/mol。sds -折叠细胞色素c在220 nm处的CD光谱仅显示部分下降（25%）。这表明，与gdnhcl变性细胞色素c完全失去螺旋结构相比，大量的螺旋结构仍然折叠。从血红素吸收和共振拉曼光谱推断，sds未折叠细胞色素c的血红素结构显示，血红素上有大量（约95%）错连接的组氨酸，这在折叠过程中起到了动力学陷阱的作用。通过时间分辨共振能量转移也监测了与细胞色素c展开相关的结构变化，结果表明，色氨酸的荧光寿命从天然蛋白的12 ps急剧增加到展开蛋白的0.63 ns，这与Trp59远离血红素移动10埃有关。荧光衰减的最大熵法分析表明，与天然蛋白中狭窄分布的构象相比，sds -未折叠的细胞色素c中有各种构象底态的生长。再折叠由三个动力学步骤组成；第一个非常快（大约8毫秒），并被分配到His26的解离，这为蛋白质走向正确的折叠途径铺平了道路。另外两个较慢的步骤可能是由链错组织和脯氨酸异构化引起的。爆发相振幅的缺失支持了这样一种观点，即在完全展开的细胞色素c折叠中观察到的爆发相对应于产生重要二级结构的分子坍塌。部分展开状态代表了折叠途径中一种独特的中间状态。
The mechanism of unfolding of ferricytochrome c induced by the surfactant sodium dodecyl sulfate has been studied by heme absorption, tryptophan fluorescence, circular dichroism, resonance Raman scattering, stopped-flow and time-resolved resonance energy transfer to obtain a comprehensive view of the whole process. Unfolding occurred at an almost specific molecular ratio of SDS/cytochrome c in the concentration range (20-50 mu M) studied here. However there appears to be a point at approximate to 0.6 mM SDS where unfolding begins to occur for lower cytochrome c concentrations. The kinetics of unfolding revealed only a single transition with a rate constant of 33 s(-1) (at 298 K, [SDS] = 8.7 mM) and activation energy barrier of approximate to 16 kJ/mol, indicating that other associated steps, if any, are too fast to be significantly populated. The free energy change (Delta G degrees) involved with the unfolding transition was estimated to be about 16.8 kJ/mol. The CD spectrum at 220 nm of SDS-unfolded cytochrome c shows only a partial decrease (25 %). indicating that a significant amount of helical structure remains folded in contrast to a complete loss of helical structure in GdnHCl-denatured cytochrome c. The heme structure in SDS-unfolded cytochrome c, as deduced from heme absorption and resonance Raman spectra, shows a major population (approximate to 95 %) of mis-ligated histidine to the heme which acts as a kinetic trap in the folding process. The structural changes associated with cytochrome c unfolding were also monitored by time-resolved resonance energy transfer which shows a drastic increase in tryptophan fluorescence lifetime from 12 ps in the native protein to 0.63 ns in the unfolded one, associated with a movement of Trp59 by 10 Angstrom away from heme. The maximum entropy method analysis of fluorescence decay indicated the growth of various conformational substates in SDS-unfolded cytochrome c in contrast to narrowly distributed conformations in the native protein. The refolding was comprised of three kinetic steps; the first was significantly fast (approximate to 8 ms) and was assigned to the dissociation of His26 that paves the protein towards correct folding pathway. The other two slower steps probably arise from chain misorganization and prolyl isomerization. The absence of a burst-phase amplitude supports the idea that the burst phase observed in the folding from completely unfolded cytochrome c corresponds to a molecular collapse that produces significant secondary structure. The partially unfolded state represents a unique intermediate state in the folding pathway.