Rates of Unfolding, Rather than Refolding, Determine Thermal Stabilities of Thermophilic, Mesophilic, and Psychrotrophic 3-Isopropylmalate Dehydrogenases

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• 作者：É ; va Gr&aacute ; czer ; Andrea Varga ; Istv&aacute ; n Hajdú ; Bogdan Melnik ; Andr&aacute ; s Szil&aacute ; gyi ; Gennady Semisotnov ; Pé ; ter Z&aacute ; vodszky ; M&aacute ; ria Vas
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：October 16, 2007
• 年：2007
• 卷：46
• 期：41
• 页码：11536 - 11549
• 全文大小：817K
• 年卷期：v.46,no.41(October 16, 2007)
• ISSN：1520-4995

文摘

The relationship between the thermal stability of proteins and rates of unfolding and refoldingis 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, andVibrio 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 ~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 reducesthe rates of unfolding; this effect is more prominent for the less stable enzyme variants. Refolding is atwo-step or multistep first-order process involving an inactive intermediate(s). The restoration of the nativestructure and reactivation take place with a half-time of a few minutes for all three IPMDHs. Thus, thecomparative experimental unfolding-refolding studies of the three IPMDHs with different thermostabilitieshave revealed a close relationship between thermostability and unfolding rate. Structural analysis hasshown that the differences in the molecular contacts between selected nonconserved residues are responsiblefor the different rates of unfolding. On the other hand, the folding rates might be correlated with theabsolute 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.