Gráczer Eva, Varga Andrea, Hajdú István, Melnik Bogdan, Szilágyi András, Semisotnov Gennady, Závodszky Péter, Vas Mária
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 7, H-1518 Budapest, Hungary.
Biochemistry. 2007 Oct 16;46(41):11536-49. doi: 10.1021/bi700754q. Epub 2007 Sep 22.
The relationship between the thermal stability of proteins and rates of unfolding and refolding is still an open issue. The data are very scarce, especially for proteins with complex structure. Here, time-dependent denaturation-renaturation experiments on Thermus thermophilus, Escherichia coli, and Vibrio sp. I5 3-isopropylmalate dehydrogenases (IPMDHs) of different heat stabilities are presented. Unfolding, as monitored by several methods, occurs in a single first-order step with half-times of approximately 1 h, several minutes, and few seconds for the thermophilic, mesophilic, and psychrotrophic enzymes, respectively. The binding of Mn*IPM (the manganese complex of 3-isopropylmalate) markedly reduces the rates of unfolding; this effect is more prominent for the less stable enzyme variants. Refolding is a two-step or multistep first-order process involving an inactive intermediate(s). The restoration of the native structure and reactivation take place with a half-time of a few minutes for all three IPMDHs. Thus, the comparative experimental unfolding-refolding studies of the three IPMDHs with different thermostabilities have revealed a close relationship between thermostability and unfolding rate. Structural analysis has shown that the differences in the molecular contacts between selected nonconserved residues are responsible for the different rates of unfolding. On the other hand, the folding rates might be correlated with the absolute contact order, which does not significantly vary between IPMDHs with different thermostabilities. On the basis of our observations, folding rates appear to be dictated by global structural characteristics (such as native topology, i.e., contact order) rather than by thermodynamic stability.
蛋白质的热稳定性与解折叠和重折叠速率之间的关系仍是一个未解决的问题。相关数据非常稀少,尤其是对于结构复杂的蛋白质。在此,我们展示了对嗜热栖热菌、大肠杆菌和弧菌属I5的不同热稳定性的3 - 异丙基苹果酸脱氢酶(IPMDHs)进行的时间依赖性变性 - 复性实验。通过多种方法监测发现,解折叠以单一的一级反应步骤进行,嗜热酶、嗜温酶和嗜冷酶的半衰期分别约为1小时、几分钟和几秒。Mn*IPM(3 - 异丙基苹果酸的锰配合物)的结合显著降低了解折叠速率;这种效应对于稳定性较差的酶变体更为显著。重折叠是一个涉及无活性中间体的两步或多步一级过程。对于所有三种IPMDHs,天然结构的恢复和再激活的半衰期为几分钟。因此,对三种具有不同热稳定性的IPMDHs进行的比较实验性解折叠 - 重折叠研究揭示了热稳定性和解折叠速率之间的密切关系。结构分析表明,选定的非保守残基之间分子接触的差异导致了解折叠速率的不同。另一方面,折叠速率可能与绝对接触顺序相关,而在具有不同热稳定性的IPMDHs之间,绝对接触顺序并没有显著变化。基于我们的观察,折叠速率似乎由整体结构特征(如天然拓扑结构,即接触顺序)决定,而非由热力学稳定性决定。