Physico-chemical and in-silico analysis of a phytocystatin purified fromBrassica junceacultivar RoAgro 5444

Abstract

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.

Insight into the biochemical characterization of phytocystatin from Glycine max and its interaction with Cd+2 and Ni+2

“…The yield is higher than the reported values of yield for other phytocystatins . The purified SBPC was found to be of molecular weight 19.05 kDa which is in the range of group II phytocystatin family members . The homogeneity of the purified SBPC was exhibited by the presence of a single band in native PAGE.…”

“…Based upon the physical properties of the SBPC, SBPC is considered to belong in the class 2 phytocystatin . Literature reports suggest that phytocystatins show higher stability over a broad range of temperature and pH . The temperature profile of the purified SBPC indicates that SBPC is stable in the temperature ranging from 30°C to 50°C.…”

“…Phytocystatins are plant‐derived cystatins which forms an independent subfamily with a molecular weight in the range of 8 to 25 kDa . Phytocystatins are further classified into three groups—Group 1 includes phytocystatins of low molecular weight (12‐16 kDa), Group 2 includes phytocystatins of 23‐kDa molecular weight that are capable of inhibiting the proteinases of Legumain (C13) family such as in case of cabbage, and high molecular weight phytocystatins with multiple tandem cystatin domains such as in case of potato and tomato are grouped in Group 3 . Phytocystatins possess highly conserved characteristic QXVXG sequence of cystatins.…”

“…Phytocystatins possess highly conserved characteristic QXVXG sequence of cystatins. In addition to the conserved sequence, a dipeptide of the proline‐tryptophan sequence is also present at the C‐terminal region along with a conserved glycine residue in the N‐terminal region . Phytocystatins play an important role in survival as well as in maintaining the healthy state of the living system.…”

Interaction of almond cystatin with pesticides: Structural and functional analysis

“…They have been earlier identified in mammalian systems and have been later on described in plants. Phytocystatins have been mainly purified from seeds but have also been detected in other plant tissues including rice, soybean, potato, barley, celosia, maize, sugarcane, alfalfa, mustard, and tobacco . Phytocystatins have dual role, as defense proteins and as endogenous regulators of protein turnover.…”