title: Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in stem bromelain near its isoelectric point.
abstract: Stem bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T(m)s), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the protein's isoelectric point.
authors: Institute of Microbial Technology (CSIR), Chandigarh, India.
authors: Sandeep
authors: S
authors: Dave
authors: Sahil
authors: S
authors: Mahajan
authors: Vemika
authors: V
authors: Chandra
authors: Pawan
authors: P
authors: Gupta
mesh_terms: D000042:Absorption / Q000187:drug effects; D020106:Acrylamide / Q000737:chemistry; D000682:Amyloid / Q000737:chemistry*; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001963:Bromelains / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D004795:Enzyme Stability / Q000187:drug effects; D019791:Guanidine / Q000494:pharmacology; D007526:Isoelectric Point; D009391:Nephelometry and Turbidimetry; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014270:Trifluoroethanol / Q000494:pharmacology*; D014364:Tryptophan / Q000378:metabolism
chemical_list: D000682:Amyloid; D000817:Anilino Naphthalenesulfonates; D020106:Acrylamide; C027132:1-anilino-8-naphthalenesulfonate; D014270:Trifluoroethanol; D014364:Tryptophan; D001963:Bromelains; C091760:stem bromelain; D019791:Guanidine