Open AccessCrystallization and preliminary characterization of a highly thermostable lectin from Trichosanthes dioica and comparison with other Trichosanthes lectins
Author(s) -
Dharkar Poorva D.,
Anuradha P.,
Gaikwad Sushama M.,
Suresh C. G.
Publication year - 2006
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s174430910600265x
Subject(s) - trichosanthes , lectin , chemistry , crystallization , dynamic light scattering , denaturation (fissile materials) , thermal stability , crystallography , urea , chromatography , nuclear chemistry , biochemistry , materials science , organic chemistry , medicine , alternative medicine , pathology , traditional chinese medicine , nanoparticle , nanotechnology
A lectin from Trichosanthes dioica seeds has been purified and crystallized using 25%( w / v ) PEG 2K MME, 0.2 M ammonium acetate, 0.1 M Tris–HCl pH 8.5 and 50 µl 0.5%( w / v ) n ‐octyl β‐ d ‐glucopyranoside as thick needles belonging to hexagonal space group P 6 4 . Unit‐cell parameters were a = b = 167.54, c = 77.42 Å. The crystals diffracted to a Bragg spacing of 2.8 Å. Both the structures of abrin‐a and T. kirilowii lectin could be used as a model in structure determination using the molecular‐replacement method; however, T. kirilowii lectin coordinates gave better values of reliability and correlation parameters. The thermal, chemical and pH stability of this lectin have also been studied. When heated, its haemagglutination activity remained unaffected up to 363 K. Other stability studies show that 4 M guanidinium hydrochloride (Gdn–HCl) initiates unfolding and that the protein is completely unfolded at 6 M Gdn–HCl. Treatment with urea resulted in a total loss of activity at higher concentrations of denaturant with no major structural changes. The protein remained stable over a wide pH range, from pH 6 to pH 12, except for partial unfolding at extremely alkaline pH. The role of disulfide bonds in the protein stability was found to be insignificant. Rayleigh light‐scattering studies showed no molecular aggregation in any of the extreme treated conditions. The unusual stability of this lectin resembles that of type II ribosome‐inactivating proteins (type II RIPs), which is also supported by structure determination. The structural features observed in a preliminary electron‐density map were compared with the other two available Trichosanthes lectin structures.