Investigation of folding unfolding process of a new variant of dihydrofolate reductase protein from Zebrafish

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• 作者：Charu Thapliyal^a ; ^b ; Neha Jain^b ; Pratima Chaudhuri (Chattopadhyay)^a ; ^{pratimachaudhuri@yahoo.com" class="auth_mail" title="E-mail the corresponding author}
• 关键词：ANS ; 8-anilino-1-naphthalene-sulfonic acid ; CD ; circular dichroism ; DHF ; dihydrofolic acid ; GdnHCl ; guanidine hydrochloride ; GSH ; reduced glutathione ; GSSG ; oxidised glutathione ; IPTG ; isopropyl- β- D-1-thiogalactopyranoside ; NADPH ; nicotinamide adenine dinucleotide phosphate ; THF ; tetrahydrofolate
• 刊名：International Journal of Biological Macromolecules
• 出版年：2016
• 出版时间：October 2016
• 年：2016
• 卷：91
• 期：Complete
• 页码：736-743
• 全文大小：1228 K

文摘

The folding and unfolding mechanisms of a small monomeric protein, Dihydrofolate reductase (1.5.1.3.) from a new variant, Zebrafish (zDHFR) has been studied through GdnHCl denaturation, followed by its refolding through dilution of the denaturant. Intrinsic and extrinsic fluorescence, far-UV CD and enzyme activity were employed to monitor structural and functional changes due to chemical denaturation. The unfolding transitions monitored by intrinsic fluorescence showed that GdnHCl based denaturation of zDHFR is reversible. At low concentration of the denaturant, zDHFR forms intermediate species as reflected by increased fluorescence intensity compared to the native and fully unfolded form. Equilibrium unfolding transition study of zDHFR induced by GdnHCl exhibited three- state process. The non- coincidence of fluorescence and far-UVCD based transitions curves support the establishment of three state model of zDHFR protein which involves native, intermediate and unfolded forms. Analysis of the equilibrium unfolding transition suggests the presence of non- native intermediate species. A comparative study of various species of DHFR shows that zDHFR has comparable thermodynamic stability with human counterpart and thus proved to be a good in vitro model system for structure- function relationship studies. Understanding various conformational states during the folding unfolding process of the zDHFR protein may provide important clues towards designing inhibitors against this important protein involved in cell cycle regulation.