title: Conformational changes during amyloid fibril formation of pancreatic thiol proteinase inhibitor: effect of copper and zinc.
abstract: Pancreatic thiol proteinase inhibitor (PTPI), a variant of cystatin superfamily of cysteine protease inhibitors, has been isolated from pancreas of Capra hircus. In the present study, we examined the effects of acid denaturation and a co-solvent on PTPI with a focus on protein conformational changes and amyloid fibril formation. The results demonstrate that PTPI can form amyloid like fibrils. Acid denaturation as studied by CD and fluorescence spectroscopy showed that PTPI populates three partly unfolded species, a native like state at pH 3.0, a structured molten globule at pH 1.0 and partly unfolded species at pH 2.0, from each of which amyloid like fibrils grow as assessed by Thioflavin T (ThT) spectroscopy. Effect of trifluoroethanol (TFE) on acid induced states of PTPI was analyzed. TFE stabilized each of the three acid-induced intermediates at predenaturational concentrations (10%) and accelerated fibril formation. Morphology of the protein species at the beginning and end of reactions was observed using transmission electron microscopy. Solvent conditions were decisive for final fibril morphology. Biometals, Cu(2+) and Zn(2+) produced a concentration dependent decline in ThT fluorescence suggesting deaggregation of the fibrils. When added prior to amyloid fibril initiation 50 μM Cu(2+) or 10 μM Zn(2+) prevented any amyloid aggregation. Implications for therapeutics in view of Cu(2+) and Zn(2+) as essential micronutrients are suggested.
authors.affiliation: Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002 UP, India.
authors.forename: Medha
authors.initials: M
authors.lastname: Priyadarshini
authors.forename: Bilqees
authors.initials: B
authors.lastname: Bano
mesh_terms: D000682:Amyloid / Q000096:biosynthesis*; D000818:Animals; D052160:Benzothiazoles; D002942:Circular Dichroism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry*; D006041:Goats / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D046529:Microscopy, Electron, Transmission; D009691:Nucleic Acid Denaturation; D010179:Pancreas / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013844:Thiazoles; D014270:Trifluoroethanol / Q000494:pharmacology
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D015853:Cysteine Proteinase Inhibitors; D013844:Thiazoles; C009462:thioflavin T; D014270:Trifluoroethanol