Structural and functional studies on a variant of cystatin purified from brain of Capra hircus

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 × 10⁻⁷ cm² s⁻¹, respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (K_i = 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.