Investigation of folding unfolding process of a new variant of dihydrofolate reductase protein from Zebrafish.
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.
Amity Institute of Biotechnology, Molecular Biophysics Laboratory, Amity University, Sector 125, Noida, Uttar Pradesh 201313, India; Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, HauzKhas, New Delhi- 110016, India.
Charu
C
Thapliyal
Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, HauzKhas, New Delhi- 110016, India.
Neha
N
Jain
Amity Institute of Biotechnology, Molecular Biophysics Laboratory, Amity University, Sector 125, Noida, Uttar Pradesh 201313, India. Electronic address: pratimachaudhuri@yahoo.com.
Pratima
P
Chaudhuri Chattopadhyay
D000818:Animals; D004795:Enzyme Stability; D006801:Humans; D000072417:Protein Domains; D058767:Protein Unfolding; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D015027:Zebrafish / Q000378:metabolism; D029961:Zebrafish Proteins / Q000737:chemistry* / Q000378:metabolism
D029961:Zebrafish Proteins; D013762:Tetrahydrofolate Dehydrogenase
CD spectroscopy of DHFR; Equilibrium unfolding of DHFR; Fluorescence spectroscopy; Refolding of DHFR; Zebrafish dihydrofolate reductase