Dave, Sandeep and Mahajan, Sahil and Chandra, Vemika and Gupta, Pawan (2011) Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in stem bromelain near its isoelectric point. International journal of biological macromolecules, 49 (4). pp. 536-42. ISSN 1879-0003
Full text not available from this repository. (Request a copy)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.
| Item Type: | Article |
|---|---|
| Additional Information: | Copyright of this article belongs to Elsevier Science |
| Uncontrolled Keywords: | Alkaline denaturation; Bromelain; Fluoroalcohol; Protein folding |
| Subjects: | Q Science > QR Microbiology |
| Depositing User: | Dr. K.P.S.Sengar |
| Date Deposited: | 09 Dec 2011 06:54 |
| Last Modified: | 09 Dec 2011 06:54 |
| URI: | http://crdd.osdd.net/open/id/eprint/459 |
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