Trifluoroethanol-induced "molten globule" state in stem bromelain.
2,2,2-Trifluoroethanol (TFE) denatures proteins but also stabilizes/induces alpha helical conformation in partially/completely unfolded proteins. As reported earlier from this laboratory, stem bromelain is known to exist as a partially folded intermediate (PFI) at pH 2.0. The effect of increasing concentration of TFE on the PFI of bromelain has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino 8-naphthalene sulfonic acid (ANS), and near-UV CD temperature transition. Far-UV CD spectra show considerable accumulation of secondary structure at 70% (v/v) concentration of TFE with spectral features resembling the pH 7.0 preparation. Interestingly the partially folded intermediate regained significant tertiary structure/interactions, with increasing concentration of TFE, and at 60% (v/v) TFE approached almost that of the pseudo native (pH 7.0) state. Further increase to 70% (v/v) TFE, however, resulted in complete loss of tertiary structure/interactions. Studies on tryptophan fluorescence also suggested the induction of some compact structure at 60% (v/v) concentration of TFE. The partially folded intermediate showed enhanced binding of the fluorescent probe (ANS) in the presence of 60% (v/v) TFE. Taken together these observations suggest a "molten globule" state between 60 and 70% (v/v) TFE. Thermal transition studies in the near-UV CD region indicated cooperative transition for PFI in the presence of 60% (v/v) TFE changing to noncooperative transition at 70% (v/v) TFE. This was accompanied by a shift in the midpoint of thermal denaturation (T(m)) from 58 to 51 degrees C. Gradual transition and loss of cooperative thermal unfolding in the 60-70% (v/v) range of TFE also support the existence of the molten globule state.
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, India.
Pawan
P
Gupta
Rizwan H
RH
Khan
M
M
Saleemuddin
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry* / Q000378:metabolism; D001963:Bromelains / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D013053:Spectrophotometry / Q000379:methods; D013696:Temperature; D014270:Trifluoroethanol / Q000494:pharmacology*; D014466:Ultraviolet Rays
D000817:Anilino Naphthalenesulfonates; D000894:Anti-Inflammatory Agents, Non-Steroidal; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D014270:Trifluoroethanol; D001963:Bromelains