Physico-chemical and in-silico analysis of a phytocystatin purified from<i>Brassica juncea</i>cultivar RoAgro 5444

Biochemistry and Cell Biology - Tập 94 Số 6 - Trang 584-596 - 2016
Shumaila Khan1, Sabahuddin Ahmad2, Mohammad Imran Siddiqi2, Bilqees Bano1
1Department of Biochemistry, Faculty of Life Science, Aligarh Muslim University, Aligarh 202002, India
2Molecular and Structural Biology Division, Council of Scientific and Industrial Research-Central Drug Research Institute (CSIR-CDRI), Lucknow 226031, Uttar Pradesh, India.

Tóm tắt

This study describes the isolation and purification of a phytocystatin from seeds of Brassica juncea (Indian mustard; cultivar RoAgro 5444), which is an important oilseed crop both agriculturally and economically. The protein was purified by gel filtration chromatography with 24.3% yield and 204-fold purification, and visualised by 2D gel electrophoresis. The 18.1 kDa mustard cystatin was highly specific for cysteine proteinases. The plant cystatin inhibited cathepsin B, confirming its role in conferring pest resistance. The inhibitor was highly stable over a pH range of 3–10 and retained significant inhibitory potential up to 70 °C. The stoichiometry of its interaction with papain, determined by isothermal calorimetry, suggests a 1:1 complex. Secondary structural elements calculated by far-UV circular dichroism (CD) spectroscopy show an 18.8% α-helical and 21% β-sheet structure. The protein was a non-competitive inhibitor of thiol proteinases. The Stokes radius and frictional co-efficient were used to describe the shape and size of the protein. Homology modelling and docking studies proposed a prototype illustrating the Brassica phytocystatin mediated papain inhibition. Molecular dynamics (MD) study revealed the excellent stability of the papain–phytocystatin complex during a simulation for 100 ns. Detailed results identify the mustard cystatin as an important member of the phytocystatin family.

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Biochemistry and Cell Biology

Tập 94 Số 6

584-596

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