title: Structural and functional studies on a variant of cystatin purified from brain of Capra hircus.
abstract: 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 Å and 8.18 × 10-7cm2s-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-1deg-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.
authors.affiliation: a Faculty of Life Sciences, Department of Biochemistry , AMU , Aligarh , Uttar Pradesh 202002 , India.
authors.forename: Peerzada Shariq Shaheen
authors.initials: PSS
authors.lastname: Khaki
authors.affiliation: a Faculty of Life Sciences, Department of Biochemistry , AMU , Aligarh , Uttar Pradesh 202002 , India.
authors.forename: Anna
authors.initials: A
authors.lastname: Feroz
authors.affiliation: b Faculty of Life Sciences, Department of Zoology , AMU , Aligarh , Uttar Pradesh 202002 , India.
authors.forename: Fakhra
authors.initials: F
authors.lastname: Amin
authors.affiliation: c Medical Microbiology and Molecular Biology Laboratory, Interdisciplinary Biotechnology Unit , AMU , Aligarh , Uttar Pradesh 202002 , India.|d Department of Pharmacognosy, College of Pharmacy , King Saud University , Riyadh 11451 , Saudi Arabia.
authors.forename: Md Tabish
authors.initials: MT
authors.lastname: Rehman
authors.affiliation: a Faculty of Life Sciences, Department of Biochemistry , AMU , Aligarh , Uttar Pradesh 202002 , India.
authors.forename: Waseem Feeroze
authors.initials: WF
authors.lastname: Bhat
authors.affiliation: a Faculty of Life Sciences, Department of Biochemistry , AMU , Aligarh , Uttar Pradesh 202002 , India.
authors.forename: Bilqees
authors.initials: B
authors.lastname: Bano
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D001963:Bromelains / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D015891:Cystatins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005361:Ficain / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006041:Goats; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010206:Papain / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000072756:Protein Conformation, alpha-Helical; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D001963:Bromelains; D010206:Papain; D005361:Ficain
keywords: caprine brain cystatin; immunodiffusion; isothermal titration calorimetry; mammalian cystatin; purification; spectroscopy; thiol proteinase inhibitor