Structural and functional studies on a variant of cystatin purified from brain of Capra hircus
| Autor: | Tabish Rehman, Anna Feroz, Bilqees Bano, Waseem Feeroze Bhat, Fakhra Amin, Peerzada Shariq Shaheen Khaki |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Protein Conformation alpha-Helical Circular dichroism Size-exclusion chromatography Cysteine Proteinase Inhibitors Substrate Specificity 03 medical and health sciences chemistry.chemical_compound Structural Biology parasitic diseases Papain Animals Ficain Molecular Biology Stokes radius Brain Chemistry Chromatography 030102 biochemistry & molecular biology Molecular mass Chemistry Goats Brain Isothermal titration calorimetry General Medicine Hydrogen-Ion Concentration Bromelains Cystatins Molecular Weight Kinetics 030104 developmental biology Thermodynamics Electrophoresis Polyacrylamide Gel Cystatin |
| Zdroj: | Journal of biomolecular structuredynamics. 35(8) |
| ISSN: | 1538-0254 |
| Popis: | Cystatins, known for their ubiquitous presence in mammalian system are thiol protease inhibitors serving important physiological functions. Here, we present a variant of cystatin isolated from brain of Capra hircus (goat) which is glycosylated but lacks disulphide bonds. Caprine brain cystatin (CBC) was isolated using alkaline treatment, ammonium sulphate fractionation (40-60%) and gel filtration chromatography on Sephacryl S-100HR column with an overall yield of 26.29% and 322-fold purification. The inhibitor gave a molecular mass of ~44 kDa as determined by SDS-PAGE and gel filtration behaviour. The Stokes radius and diffusion coefficient of CBC were 27.14 A and 8.18 × 10-7 cm2 s-1, respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (Ki = 4.10 nM) followed by ficin and bromelain. CBC possessed 34.7% α-helical content as observed by CD spectroscopy. UV, fluorescence, CD and FTIR spectroscopy revealed significant conformational change upon CBC-papain complex formation. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic parameters - ΔH, ΔS, ΔG along with N (binding stoichiometry) for CBC-papain complex formation. Binding stoichiometry (N = .97 ± .07 sites) for the CBC-papain complex indicates that cystatin is surrounded by nearly one papain molecule. Negative ΔH (-5.78 kcal mol-1) and positive ΔS (11.01 cal mol-1 deg-1) values suggest that the interaction between CBC and papain is enthalpically as well as entropically favoured process. The overall negative ΔG (-9.19 kcal mol-1) value implies a spontaneous CBC-papain interaction. |
| Databáze: | OpenAIRE |
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