Iwakura M, Jones B E, Falzone C J, Matthews C R
Department to Chemistry, Pennsylvania State University, University Park 16802.
Biochemistry. 1993 Dec 14;32(49):13566-74. doi: 10.1021/bi00212a024.
The rate-limiting steps in the folding of dihydrofolate reductase from Escherichia coli have been shown to involve the conversion of a set of four intermediates to a corresponding set of native conformers via four parallel channels [Jennings et al. (1993) Biochemistry 32, 3783-3789]. Fluorescence and absorbance studies of the unfolding and refolding of the C85S/C152E double mutant at various final urea concentrations reveal two slow folding reactions, two fewer than observed in the wild-type protein. Refolding in the presence of substoichiometric levels of the inhibitor methotrexate shows that the two remaining slow reactions correspond to two parallel channels which lead to a pair of native conformers capable of binding the inhibitor. A combination of stopped-flow circular dichroism and cofactor binding studies confirms that the four parallel channels observed in the wild-type protein have collapsed into two channels in the mutant. Kinetic and equilibrium studies of the single cysteine mutants suggest that replacements of Cysteine-85 which perturb the hydrophobic core containing this side chain are responsible for the simplification of the kinetic mechanism. These results demonstrate that at least two of the parallel folding channels in dihydrofolate reductase arise when tertiary structure develops and are not dependent upon cis/trans isomerization at prolyl peptide bonds.
来自大肠杆菌的二氢叶酸还原酶折叠过程中的限速步骤已被证明涉及一组四个中间体通过四个平行通道转化为相应的一组天然构象体[詹宁斯等人（1993年），《生物化学》32卷，3783 - 3789页]。对C85S/C152E双突变体在不同最终尿素浓度下的解折叠和再折叠进行的荧光和吸光度研究揭示了两个缓慢的折叠反应，比野生型蛋白中观察到的少两个。在亚化学计量水平的抑制剂甲氨蝶呤存在下进行再折叠表明，剩下的两个缓慢反应对应于两个平行通道，这两个通道通向一对能够结合抑制剂的天然构象体。停流圆二色性和辅因子结合研究的结合证实，在野生型蛋白中观察到的四个平行通道在突变体中已合并为两个通道。对单个半胱氨酸突变体的动力学和平衡研究表明，扰动包含该侧链的疏水核心的半胱氨酸-85的取代是动力学机制简化的原因。这些结果表明，二氢叶酸还原酶中至少两个平行折叠通道是在三级结构形成时出现的，并且不依赖于脯氨酰肽键处的顺/反异构化。
