Crystallization and preliminary characterization of a highly thermostable lectin from Trichosanthes dioica and comparison with other Trichosanthes lectins.
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 microl 0.5%(w/v) n-octyl beta-D-glucopyranoside as thick needles belonging to hexagonal space group P6(4). Unit-cell parameters were a = b = 167.54, c = 77.42 A. The crystals diffracted to a Bragg spacing of 2.8 A. 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.
Dharkar
Poorva D
PD
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India.
Anuradha
P
P
Gaikwad
Sushama M
SM
Suresh
C G
CG
D003460:Crystallization; D018360:Crystallography, X-Ray; D019791:Guanidine / Q000494:pharmacology; D006384:Hemagglutination; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D037121:Plant Lectins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D012639:Seeds / Q000737:chemistry*; D029778:Trichosanthes / Q000737:chemistry*; D014508:Urea / Q000494:pharmacology
D037121:Plant Lectins; D014508:Urea; D019791:Guanidine