Structural characterization and low-resolution model of BJ-48, a thrombin-like enzyme from Bothrops jararacussu venom: Evidence of pH effect on hydrodynamic properties.
Herbert L. M. Guedes1; Floriano P. Silva-Jr1; Giani Alexandre1; Carlos Correa Netto1,3; Cristiane M. C. de Salles1;Cristiano L. P. de Oliveira4; Íris Torriani4; Salvatore Giovanni De Simone1,2
1 Laboratório de Bioquímica de Proteínas e Peptídeos, Departamento de Bioquímica e Biologia Molecular, IOC, FIOCRUZ, Rio de Janeiro, Brazil.
2 Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Brazil.
3 Instituto Vital Brazil, Niterói, Brazil.
4 Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Campinas, Brazil.
Thrombin-like enzymes (TLEs) are important components of snake venoms due to their involvement in coagulopathies occurring on envenoming. Structural characterization of this group of serine proteases is of utmost importance for better understanding their unique properties. However, the high carbohydrate content of some members of this group prevents successful crystallization for structural determination. Circumventing this difficulty, the structure of BJ-48, a highly glycosylated TLE from Bothrops jararacussu venom, was studied in solution. In pH 8.0, where the enzyme displays maximum activity, BJ-48 has a radius of gyration (Rg) value of 37Å and a maximum dimension (Dmax) value of 130Å by SAXS and a Stokes radius (SR) of 50Å by DLS. At the naturally more acidic pH (6.0) of the B. jararacussu venom BJ-48 particle behaves as a more compact particle as evidenced by SAXS (Rg = 27.9Å and Dmax = 82Å) and DLS (SR = 30Å) data. In addition, Kratky plot analysis indicates a rigid shape at pH 8.0 and a flexible shape at pH 6.0. In the other hand, center of mass of intrinsic fluorescence was not changed varying pH, possibly indicating the absence of fluorescent amino acids in the regions affect by pH variation. CD experiments carried out with BJ-48 indicate a substantially random coiled secondary structure that is not affected by pH . Low resolution model of BJ-48 presented a prolate elongated shape at pH 8.0 and a U-shape at 6.0. BJ-48 tertiary structure at pH 6.0 was maintained on heating up to 52 oC and was completely lost at 70oC. BJ-48 showed no modification on its shape after complex formation with the inhibitor benzamidine. We believe in existence of two pH-induced folding states for BJ-48, important for the biological role and stability of this enzyme.
Support: CNPq, FAPERJ, FIOCRUZ
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