RESUMO
The clostridial collagenases G and H are multidomain proteins. For collagen digestion, the domain arrangement is likely to play an important role in collagen binding and hydrolysis. In this study, the full-length collagenase H protein from Clostridium histolyticum was expressed in Escherichia coli and purified. The N-terminal amino acid of the purified protein was Ala31. The expressed protein showed enzymatic activity against azocoll as a substrate. To investigate the role of Ca(2+) in providing structural stability to the full-length collagenase H, biophysical measurements were conducted using the recombinant protein. Size exclusion chromatography revealed that the Ca(2+) chelation by EGTA induced interdomain conformational changes. Dynamic light scattering measurements showed an increase in the percent polydispersity as the Ca(2+) was chelated, suggesting an increase in protein flexibility. In addition to these conformational changes, differential scanning fluorimetry measurements revealed that the thermostability was decreased by Ca(2+) chelation, in comparison with the thermal melting point (T(m)). The melting point changed from 54 to 49°C by the Ca(2+) chelation, and it was restored to 54°C by the addition of excess Ca(2+). These results indicated that the interdomain flexibility and the domain arrangement of full-length collagenase H are reversibly regulated by Ca(2+).
Assuntos
Cálcio/metabolismo , Clostridium histolyticum/enzimologia , Íons/metabolismo , Colagenase Microbiana/química , Compostos Azo/metabolismo , Cromatografia em Gel , Clonagem Molecular , Colágeno/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Fluorometria , Expressão Gênica , Colagenase Microbiana/genética , Colagenase Microbiana/metabolismo , Conformação Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Temperatura de TransiçãoRESUMO
The identification of altruists based on non-verbal cues might offer a solution to the problem of subtle cheating. Previous studies have indicated that the ability to discriminate altruists from non-altruists emerges during evolution. However, behavioural differences with regard to social exchanges involving altruists and non-altruists have not been studied. We investigated differences in responses to videotaped altruists and non-altruists with the Faith Game. Participants tended to entrust real money to altruists more than to non-altruists, providing strong evidence that cognitive adaptations evolve as counter-strategies to subtle cheating.
