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 Ca2+ in providing structural stability to the full-length collagenase H, biophysical measurements were conducted using the recombinant protein. Size exclusion chromatography revealed that the Ca2+ chelation by EGTA induced interdomain conformational changes. Dynamic light scattering measurements showed an increase in the percent polydispersity as the Ca2+ 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 Ca2+ chelation, in comparison with the thermal melting point (Tm). The melting point changed from 54 to 49°C by the Ca2+ chelation, and it was restored to 54°C by the addition of excess Ca2+. These results indicated that the interdomain flexibility and the domain arrangement of full-length collagenase H are reversibly regulated by Ca2+.
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