Crystallization and preliminary crystallographic analysis of SMase I, a sphingomyelinase from Loxosceles laeta spider venom.
SMase I, a 32 kDa sphingomyelinase found in Loxosceles laeta venom, is responsible for the major pathological effects of spider envenomation. This toxin has been cloned and functionally expressed as a fusion protein containing a 6 x His tag at its N-terminus to yield a 33 kDa protein [Fernandes-Pedrosa et al. (2002), Biochem. Biophys. Res. Commun. 298, 638-645]. The recombinant protein possesses all the biological properties ascribed to the whole L. laeta venom, including dermonecrotic and complement-dependent haemolytic activities. Dynamic light-scattering experiments conducted at 291 K demonstrate that the sample possesses a monomodal distribution, with a hydrodynamic radius of 3.57 nm. L. laeta SMase I was crystallized by the hanging-drop vapour-diffusion technique using the sparse-matrix method. Single crystals were obtained using a buffer solution consisting of 0.08 M HEPES and 0.9 M trisodium citrate, which was titrated to pH 7.5 using 0.25 M sodium hydroxide. Complete three-dimensional diffraction data were collected to 1.8 angstroms at the Laboratório Nacional de Luz Síncrotron (LNLS, Campinas, Brazil). The crystals belong to the hexagonal system (space group P6(1) or P6(5)), with unit-cell parameters a = b = 140.6, c = 113.6 angstroms. A search for heavy-atom derivatives has been initiated and elucidation of the crystal structure is currently in progress.
Zela
S P
SP
Department of Physics, IBILCE/UNESP, CP 136, São José do Rio Preto, SP, CEP 15054-000, Brazil.
Fernandes Pedrosa
M F
MF
Murakami
M T
MT
De Andrade
S A
SA
Arni
R K
RK
Tambourgi
D V
DV
D000818:Animals; D002951:Citrates / Q000494:pharmacology; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D008027:Light; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D000077559:Sodium Citrate; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry*; D013111:Spider Venoms / Q000737:chemistry*; D013112:Spiders; D013696:Temperature
D002951:Citrates; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013111:Spider Venoms; D000077559:Sodium Citrate; D013108:Sphingomyelin Phosphodiesterase; C495583:sphingomyelinase I, Loxosceles laeta