Hydrodynamic Properties and Quaternary Structure of the 90 kDa Heat-Shock Protein: Effects of Divalent Cations
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- 作者：Cyrille Garnier ; Pascale Barbier ; Franç ; ois Devred ; German Rivas ; and Vincent Peyrot
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：October 1, 2002
- 年：2002
- 卷：41
- 期：39
- 页码：11770 - 11778
- 全文大小：152K
- 年卷期：v.41,no.39(October 1, 2002)
- ISSN：1520-4995
文摘
The 90 kDa heat-shock protein (Hsp90) is one of the major stress proteins whose overallstructure remains unknown. In this study, we investigated the influence of divalent cations Mg2+ andCa2+ on the hydrodynamic properties and quaternary structure of Hsp90. Using analytical ultracentrifugation,size-exclusion chromatography, and polyacrylamide gel electrophoresis, we showed that native Hsp90was mostly dimeric. The Hsp90 dimer had a sedimentation coefficient, sw,20
, of 6.10 ± 0.03 S, whichslightly deviated from the hydrodynamics of a globular protein. Using chemical cross-linking and analyticalultracentrifugation, we showed that Mg2+ and Ca2+ induced a tertiary conformational change of Hsp90,leading to a self-association process. In the presence of divalent cations, Hsp90 existed as a mixture ofmonomers, dimers, and tetramers at equilibrium. Finally, to identify Hsp90 domains involved in thisdivalent cation-dependent self-association, we studied the oligomerization state of the N-terminal (positions1-221) of Hsp90, the influence of an N-terminal specific ligand, geldanamycin (GA), and the effect ofC-terminal truncation on the ability of Hsp90 to oligomerize in the presence of divalent cations. Wepreviously showed that GA inhibits Hsp90 heat-induced oligomerization [Garnier, C., Protasevich, I.,Gilli, R., Tsvetkov, P., Lobachov, V., Peyrot, V., Briand, C., and Makarov, A. (1998) Biochem. Biophys.Res. Commun. 249, 197-201], but now we observed that GA does not influence divalent cation-dependentoligomerization of Hsp90, suggesting another mechanism. This mechanism involved the C-terminal partof the protein since C-terminally truncated Hsp90 did not oligomerize in the presence of divalent cations.
, of 6.10 ± 0.03 S, whichslightly deviated from the hydrodynamics of a globular protein. Using chemical cross-linking and analyticalultracentrifugation, we showed that Mg2+ and Ca2+ induced a tertiary conformational change of Hsp90,leading to a self-association process. In the presence of divalent cations, Hsp90 existed as a mixture ofmonomers, dimers, and tetramers at equilibrium. Finally, to identify Hsp90 domains involved in thisdivalent cation-dependent self-association, we studied the oligomerization state of the N-terminal (positions1-221) of Hsp90, the influence of an N-terminal specific ligand, geldanamycin (GA), and the effect ofC-terminal truncation on the ability of Hsp90 to oligomerize in the presence of divalent cations. Wepreviously showed that GA inhibits Hsp90 heat-induced oligomerization [Garnier, C., Protasevich, I.,Gilli, R., Tsvetkov, P., Lobachov, V., Peyrot, V., Briand, C., and Makarov, A. (1998) Biochem. Biophys.Res. Commun. 249, 197-201], but now we observed that GA does not influence divalent cation-dependentoligomerization of Hsp90, suggesting another mechanism. This mechanism involved the C-terminal partof the protein since C-terminally truncated Hsp90 did not oligomerize in the presence of divalent cations.