Persistent Conformational Heterogeneity of Triosephosphate Isomerase: Separation and Characterization of Conformational Isomers in Solution
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- 作者：Vitor Hugo Moreau ; Alex W. M. Rietveld ; and Sergio T. Ferreira
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：December 23, 2003
- 年：2003
- 卷：42
- 期：50
- 页码：14831 - 14837
- 全文大小：96K
- 年卷期：v.42,no.50(December 23, 2003)
- ISSN：1520-4995
文摘
Subunit dissociation of dimeric rabbit muscle triosephosphate isomerase (TIM) by hydrostaticpressure has previously been shown not to follow the expected dependence on protein concentration[Rietveld and Ferreira (1996) Biochemistry 35, 7743-7751]. This anomalous behavior was attributed topersistent conformational heterogeneity (i.e., the coexistence of long-lived conformational isomers) inthe ensemble of TIM dimers. Here, we initially show that subunit dissociation/unfolding of TIM byguanidine hydrochloride (GdnHCl) also exhibits an anomalous dependence on protein concentration.Dissociation/unfolding of TIM by GdnHCl was investigated by intrinsic fluorescence and circular dichroismspectroscopies and was found to be a highly cooperative transition in which the tertiary and secondarystructures of the protein were concomitantly lost. A procedure based on size-exclusion chromatographyin the presence of intermediate (0.6 M) GdnHCl concentrations was developed to isolate two conformationalisomers of TIM that exhibit significantly different stabilities and kinetics of unfolding by GdnHCl. Completeunfolding of the two isolated conformers at a high GdnHCl concentration (1.5 M), followed by refoldingby removal of the denaturant, completely abolished the differences in their unfolding kinetics. Theseresults indicate that such differences stem from conformational heterogeneity of TIM and are not relatedto any chemical modification of the protein. Furthermore, they add support to the notion that long-livedconformational isomers of TIM coexist in solution and provide a basis for the interpretation of the persistentheterogeneity of this protein.