Domain Structure and Denaturation of a Dimeric Mip-like Peptidyl-Prolyl cis鈥搕rans Isomerase from Escherichia coli

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• 作者：Biswanath Jana ; Amitava Bandhu ; Rajkrishna Mondal ; Anindya Biswas ; Keya Sau ; Subrata Sau
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：February 14, 2012
• 年：2012
• 卷：51
• 期：6
• 页码：1223-1237
• 全文大小：709K
• 年卷期：v.51,no.6(February 14, 2012)
• ISSN：1520-4995

文摘

FKBP22, a protein expressed by Escherichia coli, possesses PPIase (peptidyl-prolyl cis-trans isomerase) activity, binds FK506 (an immunosuppressive drug), and shares homology with Legionella Mip (a virulence factor) and its related proteins. To understand the domain structure and the folding鈥搖nfolding mechanism of Mip-like proteins, we investigated a recombinant E. coli FKBP22 (His-FKBP22) as a model protein. Limited proteolysis indicated that His-FKBP22 harbors an N-terminal domain (NTD), a C-terminal domain (CTD), and a long flexible region linking the two domains. His-FKBP22, NTD⁺ (NTD with the entire flexible region), and CTD⁺ (CTD with a truncated flexible region) were unfolded by a two-state mechanism in the presence of urea. Urea induced the swelling of dimeric His-FKBP22 molecules at the pretransition state but dissociated it at the early transition state. In contrast, guanidine hydrochloride (GdnCl)-induced equilibrium unfolding of His-FKBP22 or NTD⁺ and CTD⁺ seemed to follow three-step and two-step mechanisms, respectively. Interestingly, the intermediate formed during the unfolding of His-FKBP22 with GdnCl was not a molten globule but was thought to be composed of the partially unfolded dimeric as well as various multimeric His-FKBP22 molecules. Dimeric His-FKBP22 did not dissociate gradually with increasing concentrations of GdnCl. Very low GdnCl concentrations also had little effect on the molecular dimensions of His-FKBP22. Unfolding with either denaturant was found to be reversible, as refolding of the unfolded His-FKBP22 completely, or nearly completely, restored the structure and function of the protein. Additionally, denaturation of His-FKBP22 appeared to begin at the CTD⁺.