Trifluoroethanol-induced "molten globule" state in stem bromelain

Area of Science:

• Protein denaturation and stabilization
• Biophysical chemistry
• Enzyme structure-function relationships

Background:

• Stem bromelain exists as a partially folded intermediate (PFI) at pH 2.0.
• 2,2,2-Trifluoroethanol (TFE) is known to denature proteins and induce alpha-helical structures.

Purpose of the Study:

• To investigate the effect of increasing 2,2,2-Trifluoroethanol (TFE) concentrations on the partially folded intermediate (PFI) of stem bromelain.
• To characterize the structural changes and identify potential intermediate states.

Main Methods:

• Circular dichroism (CD) spectroscopy (far-UV and near-UV)
• Fluorescence emission spectroscopy
• Hydrophobic dye (ANS) binding assays
• Thermal denaturation studies (T(m))

Main Results:

• Far-UV CD showed increased secondary structure at 70% TFE, resembling the native state.
• Tertiary structure and interactions were regained at 60% TFE, approaching the native state.
• Tryptophan fluorescence and ANS binding indicated a compact structure at 60% TFE.
• Near-UV CD thermal transitions shifted from cooperative to noncooperative between 60% and 70% TFE.

Conclusions:

• Stem bromelain PFI transitions to a molten globule state between 60% and 70% TFE.
• This molten globule state is characterized by regained secondary and tertiary structure with altered cooperative thermal unfolding.
• TFE concentration is critical in modulating protein folding intermediates and conformational states.