PROBING THE FOLDING MECHANISM OF A LEUCINE-ZIPPER PEPTIDE BY STOPPED-FLOW CIRCULAR-DICHROISM SPECTROSCOPY
PROBING THE FOLDING MECHANISM OF A LEUCINE-ZIPPER PEPTIDE BY STOPPED-FLOW CIRCULAR-DICHROISM SPECTROSCOPY
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DOI：
10.1021/bi00039a042
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发表时间：
1995-10-03
期刊：
影响因子：
2.9
通讯作者：
MATTHEWS, CR
中科院分区：
文献类型：
--
作者：
ZITZEWITZ, JA;BILSEL, O;MATTHEWS, CR
亮氨酸拉链肽为研究控制蛋白质折叠的分子内和分子间相互作用提供了简单的模型系统。合成的 33 残基肽 GCN4-p1 源自酵母转录激活剂 GCN4，形成稳定的双分子卷曲螺旋结构 [O'Shea, E. K., Klemm, J. D., Kim, P. S., & Alber, T. (1991) Science 254, 539-544]。当适合涉及天然二聚体和未折叠单体的双态模型时，盐酸胍在 5 摄氏度和 pH 7.0 下诱导该肽的平衡解折叠，产生 10.49 +/- 0.23 kcal（梅尔二聚体）(-1) 的标准态自由能。通过停流圆二色光谱监测 GCN4-p1 的解折叠和重折叠动力学，作为肽浓度和最终变性剂浓度的函数。展开动力学表现出单指数行为，与单分子反应一致。依赖于浴肽和胍浓度的重折叠动力学可以通过简单的双分子缔合反应得到很好的描述。将所有解折叠和重折叠动力学数据同时拟合到模型 N-2 可逆箭头(ku) 2U，在不存在变性剂的情况下分别产生 4.2 x 10(5) M(-1) s(-1) 和(kf) 3.3 x 10(-3) s(-1) 的重折叠和解折叠速率常数。根据这些速率常数可以准确预测平衡展开曲线，为二态动力学模型的有效性提供了进一步的支持。
Leucine zipper peptides provide simple model systems for studying both the intramolecular and intermolecular interactions that govern protein folding. The synthetic 33-residue peptide GCN4-p1, derived from the yeast transcriptional activator GCN4, forms a stable bimolecular coiled-coil structure [O'Shea, E. K., Klemm, J. D., Kim, P. S., & Alber, T. (1991) Science 254, 539-544]. The guanidineHC1 induced equilibrium unfolding of this peptide at 5 degrees C and pH 7.0 yields a standard state free energy of 10.49 +/- 0.23 kcal (mel dimer)(-1) when fit to a two-state model involving the native dimer and the unfolded monomer. The unfolding and refolding kinetics of GCN4-p1 were monitored by stopped-flow circular dichroism spectroscopy as a function of both peptide concentration and final denaturant concentration. The unfolding kinetics displayed single-exponential behavior, consistent with a unimolecular reaction. The refolding kinetics, which are dependent on bath peptide and guanidine concentration, are well described by a simple bimolecular association reaction. A simultaneous fit of all of the unfolding and refolding kinetic data to the model, N-2 reversible arrow(ku) 2U, yields refolding and unfolding rate constants in the absence of denaturant of 4.2 x 10(5) M(-1) s(-1) and(kf) 3.3 x 10(-3) s(-1), respectively. The equilibrium unfolding curve is accurately predicted from these rate constants, providing further support for the validity of the two-state kinetic model.