Enhancement of the structural stability of full-length clostridial collagenase by calcium ions.
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+).
Faculty of Pharmacy, Iwaki Meisei University, Fukushima, Japan.
Naomi
N
Ohbayashi
Noriko
N
Yamagata
Masafumi
M
Goto
Kimiko
K
Watanabe
Youhei
Y
Yamagata
Kazutaka
K
Murayama
D001391:Azo Compounds / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D047009:Hathewaya histolytica / Q000201:enzymology*; D003094:Collagen / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005470:Fluorometry; D015870:Gene Expression; D007477:Ions / Q000378:metabolism*; D003012:Microbial Collagenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D044366:Transition Temperature
D001391:Azo Compounds; D007477:Ions; D011994:Recombinant Proteins; C028470:Azocoll; D003094:Collagen; D003012:Microbial Collagenase; D002118:Calcium