Structure and stability of an early folding intermediate of Escherichia coli trp aporepressor measured by far-UV stopped-flow circular dichroism and 8-anilino-1-naphthalene sulfonate binding.
Structure and stability of an early folding intermediate of Escherichia coli trp aporepressor measured by far-UV stopped-flow circular dichroism and 8-anilino-1-naphthalene sulfonate binding.
复制标题
通过远紫外停流圆二色性和 8-苯胺基-1-萘磺酸盐结合测量大肠杆菌 trp 阻压剂早期折叠中间体的结构和稳定性。
DOI：
10.1021/bi00071a002
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发表时间：
1993
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：
Mann,CJ;Matthews,CR
摘要：用停流远紫外圆二色谱法和8-苯胺基-1-萘磺酸荧光光谱法研究了大肠杆菌去辅阻遏蛋白的复性动力学。在混合的死时间（4-5 ms）内分别观察到二级结构和疏水表面的发展的显着增益。这些初始增加，或突发阶段振幅，绘制为最终尿素浓度的函数，表现出S形，一致的展开过渡曲线。转变曲线符合两态模型，在没有变性剂的情况下，得到的折叠自由能相似（~ 3.3 kcal/mol）。随后的三个缓慢重折叠阶段表现出与先前观察到的色氨酸荧光类似的弛豫时间和幅度[Gittelman，M.美国，和马修斯，CR（1990）生物化学29，7011-7021]。这些结果支持的建议，一个稳定的，单体的中间体是快速形成的trp脱辅基阻遏物的折叠过程中，这种物种包含了大量的二级结构和疏水表面。这个早期的中间产物经过折叠和结合反应，最终形成剩余的二级、三级和四级结构，寡聚蛋白质中发现的四级结构给确定氨基酸序列引导快速有效折叠形成天然构象的机制的问题增加了另一层复杂性。这种高级结构的发展提出了单体蛋白质折叠中没有遇到的几个问题：（1）单个亚基在结合之前获得结构吗？(2)如果亚基折叠，在什么时间范围内发生，结构是否与天然构象相似？(3)亚基之间的结合反应发生在什么阶段？以及（4）在关联事件之后折叠是否继续？
Revised Manuscript Received March 9, 1993 abstract: The refolding kinetics of Escherichia colitrp aporepressor were monitored using stopped-flow far-ultraviolet circular dichroismand 8-anilino-1-naphthalene sulfonate fluorescence spectroscopy. Significant gains in secondary structure and the development of hydrophobic surface, respectively, were observed within the dead time of mixing (4-5 ms). These initial increases, or burst phase amplitudes, plotted as a function of final urea concentration, exhibited sigmoidal, coincident unfolding transition curves. The transition curves were fit to a two-state model, and the resulting free energies of foldingin the absence of denaturant were found to be similar (~ 3.3 kcal/mol). Three subsequent slow refolding phases exhibited relaxation times and amplitudessimilar to those previously observed for tryptophan fluorescence [Gittelman, M. S., & Matthews, CR (1990) Biochemistry 29, 7011-7021]. These results support the proposals that a stable, monomeric intermediate is rapidly formed during the folding of trp aporepressor and that this species contains a significant amount of secondary structure and hydrophobic surface. This early intermediate is then processed through folding and association reactions that result in the formation of the remaining secondary, tertiary, and quaternary structure.The quaternary structure found in oligomeric proteins adds another layer of complexity to the problem of determining the mechanism by which the amino acid sequence directs the rapid and efficient folding to the nativeconformation. The devel-opment of this higher order structure raises several issues that are not encountered in the folding of monomeric proteins:(1) Do the individual subunits acquire structure prior to asso-ciation?(2) If the subunits do fold, in what time range does this happen and is the structure similar to that in the native conformation?(3) At what stage does the association reaction-(s) between subunits occur? and (4) Does folding continue after the association event?