RESUMO
Misfolding of mutant enzymes may play an important role in the pathogenesis of cystathionine beta-synthase (CBS) deficiency. We examined properties of a series of 27 mutant variants, which together represent 70% of known alleles observed in patients with homocystinuria due to CBS deficiency. The median amount of SDS-soluble mutant CBS polypeptides in the pellet after centrifugation of bacterial extracts was increased by 50% compared to the wild type. Moreover, mutants formed on average only 12% of tetramers and their median activity reached only 3% of the wild-type enzyme. In contrast to the wild-type CBS about half of mutants were not activated by S-adenosylmethionine. Expression at 18 degrees C substantially increased the activity of five mutants in parallel with increasing the amounts of tetramers. We further analyzed the role of solvent accessibility of mutants as a determinant of their folding and activity. Buried mutations formed on average less tetramers and exhibited 23 times lower activity than the solvent exposed mutations. In summary, our results show that topology of mutations predicts in part the behavior of mutant CBS, and that misfolding may be an important and frequent pathogenic mechanism in CBS deficiency.
Assuntos
Cistationina beta-Sintase/genética , Homocistinúria/genética , Mutação , Western Blotting , Catálise , Domínio Catalítico/genética , Temperatura Baixa , Cistationina beta-Sintase/química , Cistationina beta-Sintase/deficiência , Estabilidade Enzimática , Escherichia coli/genética , Homocistinúria/enzimologia , Humanos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Dobramento de Proteína , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , SolubilidadeRESUMO
Multimolecular micelles, formed by polystyrene-block-poly(methacrylic acid) in water, are studied by lattice Monte Carlo method. Electrostatic interactions are calculated in the mean-field approximation by solving the Poisson-Boltzmann equation. The model is parametrized according to available experimental data. The dependence of micellar size on pH and ionic strength is calculated and compared with experimental data. A special attention is devoted to the behavior in solutions with a low ionic strength.