The Binding of Bis-ANS to the Isolated GroEL Apical Domain Fragment Induces the Formation of a Folding Intermediate with Increased Hydrophobic Surface Not Observed in Tetradecameric GroEL

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• 作者：Alison L. Smoot ; Markandeswar Panda ; Bill T. Brazil ; Ashley M. Buckle ; Alan R. Fersht ; and Paul M. Horowitz
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：April 10, 2001
• 年：2001
• 卷：40
• 期：14
• 页码：4484 - 4492
• 全文大小：96K
• 年卷期：v.40,no.14(April 10, 2001)
• ISSN：1520-4995

文摘

The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domainfragment of GroEL, or minichaperone (residues 191-345), was investigated and compared with that ofthe GroEL tetradecamer. Although a total of seven molecules of bis-ANS bind cooperatively to thisminichaperone, most of the hydrophobic sites were induced following initial binding of one to two moleculesof probe. From the equilibrium and kinetics studies at low bis-ANS concentrations, it is evident that thenative apical domain is converted to an intermediate conformation with increased hydrophobic surfaces.This intermediate binds additional bis-ANS molecules. Tyrosine fluorescence detected denaturationdemonstrated that bis-ANS can destabilize the apical domain. The results from (i) bis-ANS titrations, (ii)urea denaturation studies in the presence and absence of bis-ANS, and (iii) intrinsic tyrosine fluorescencestudies of the apical domain are consistent with a model in which bis-ANS binds tightly to the intermediatestate, relatively weakly to the native state, and little to the denatured state. The results suggest that theconformational changes seen in apical domain fragments are not seen in the intact GroEL oligomer dueto restrictions imposed by connections of the apical domain to the intermediate domain and suppressionof movement due to quaternary structure.