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

July 28, 2017

Title

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

Author

Charu Thapliyal, Neha Jain, Pratima Chaudhuri (Chattopadhyay)

Year

2016

Journal

International Journal of Biological Macromolecules

Abstract

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.

Instrument

J-815

Keywords

Circular dichroism, Secondary structure, Protein denaturation, Protein folding, Thermodynamics, Biochemistry