Satumba W John, Mossing Michael C
Department of Chemistry and Biochemistry, University of Mississippi, University, Mississippi 38677, USA.
Biochemistry. 2002 Dec 3;41(48):14216-24. doi: 10.1021/bi026777h.
Cro binds to operator sites in lambda DNA as a dimer. Dimerization of this small repressor protein is weak, however, and proline residues in the dimer interface suggest that folding and assembly of active repressors may be complex. Cro and selected variants have been studied by circular dichroism and fluorescence. Fluorescent probes include a unique tryptophan residue in the dimer interface and extrinsic resonance energy transfer probes that monitor dimerization. Both folding and unfolding are characterized by two distinct kinetic phases. Fast processes that are complete within the 5-10 ms dead time of stopped flow experiments account for the majority of the change in the CD signal and abrupt changes in both tryptophan fluorescence and energy transfer. The slow phases show all the hallmarks of proline isomerization. The rates of the slow phases are between 0.005 and 0.02 s(-1), are relatively independent of protein and denaturant concentration, display activation energies of 20 kcal/mol, and are accelerated by the peptidyl-prolyl isomerase SlyD. Although CD measurements indicate that more than 70% of the secondary structure is regained in the refolding burst phase, intermolecular fluorescence resonance energy transfer experiments indicate that less than 25% of these subunits are assembled into dimers. Full folding and dimerization requires isomerization of the non-native prolyl isomers over hundreds of seconds.
Cro以二聚体形式与λ噬菌体DNA中的操纵位点结合。然而，这种小阻遏蛋白的二聚化作用较弱，并且二聚体界面中的脯氨酸残基表明活性阻遏蛋白的折叠和组装可能很复杂。已通过圆二色性和荧光对Cro及选定的变体进行了研究。荧光探针包括二聚体界面中一个独特的色氨酸残基以及监测二聚化的外在共振能量转移探针。折叠和去折叠均具有两个不同的动力学阶段。在停流实验5 - 10毫秒的死时间内完成的快速过程占圆二色性信号变化以及色氨酸荧光和能量转移突然变化的大部分。慢相显示出脯氨酸异构化的所有特征。慢相的速率在0.005至0.02 s⁻¹之间，相对独立于蛋白质和变性剂浓度，显示出20千卡/摩尔的活化能，并被肽基脯氨酰异构酶SlyD加速。尽管圆二色性测量表明在复性爆发阶段超过70%的二级结构得以恢复，但分子间荧光共振能量转移实验表明这些亚基中不到25%组装成了二聚体。完全折叠和二聚化需要非天然脯氨酰异构体在数百秒内进行异构化。
