Herbst R, Gast K, Seckler R
Universität Regensburg, Institut für Biophysik und Physikalische Biochemie, D-93040 Regensburg, Germany.
Biochemistry. 1998 May 5;37(18):6586-97. doi: 10.1021/bi972928i.
The guanidine-induced unfolding of firefly (Photinus pyralis) luciferase involves two inactive equilibrium intermediates and is freely reversible at low protein concentration and low temperature. However, reactivation is exceedingly slow so that the equilibrium is attained only after several days of incubation and reactivation yields decrease strongly with increasing protein concentration, suggesting that aggregation is a competing side reaction [Herbst et al. (1997) J. Biol. Chem. 272, 7099-7105]. We investigated the role of the equilibrium intermediates in the aggregation process using size-exclusion chromatography and dynamic light scattering to monitor their association state. Although the more unfolded intermediate aggregated much more rapidly, both intermediates associated irreversibly without a conformational change visible by fluorescence or circular dichroism, forming small oligomers which remained soluble in the presence of the denaturant. The association kinetics are compatible with a nucleated polymerization mechanism. Unfolding kinetics at 1 M denaturant indicated the presence of a further inactive intermediate capable to reactivate rapidly with kinetics similar to those observed for luciferase reactivation in the presence of cell extracts. The data suggest a kinetic trap in luciferase refolding that is accessible from both equilibrium intermediate conformations and is avoided in the presence of molecular chaperones.
胍诱导的萤火虫（Photinus pyralis）荧光素酶的去折叠涉及两个无活性的平衡中间体，并且在低蛋白浓度和低温下是完全可逆的。然而，重新激活极其缓慢，以至于只有在孵育几天后才能达到平衡，并且随着蛋白浓度的增加，重新激活产率急剧下降，这表明聚集是一个竞争性的副反应[Herbst等人（1997年）《生物化学杂志》272卷，7099 - 7105页]。我们使用尺寸排阻色谱和动态光散射来监测它们的缔合状态，研究了平衡中间体在聚集过程中的作用。尽管更去折叠的中间体聚集得更快，但两种中间体都不可逆地缔合，且没有荧光或圆二色性可见的构象变化，形成了小的寡聚体，这些寡聚体在变性剂存在下仍可溶。缔合动力学与成核聚合机制相符。在1 M变性剂下的去折叠动力学表明存在另一种无活性中间体，它能够以类似于在细胞提取物存在下观察到的荧光素酶重新激活的动力学快速重新激活。数据表明荧光素酶重折叠中存在一个动力学陷阱，它可从两种平衡中间体构象进入，并且在分子伴侣存在时可避免。
