Abstract
Spectroscopic, calorimetric, and proteolytic methods were utilized to evaluate the stability of the kinetically stable, differentially glycosylated, dimeric serine protease milin as a function of pH (1.0–11.0), temperature, urea, and GuHCl denaturation in presence of 8 M urea at pH 2.0. The stability of milin remains equivalent to that of native at pH 1.0–11.0. However, negligible and reversible alteration in structure upon temperature transition has been observed at pH 2.0 and with 1.6 M GuHCl. Irreversible and incomplete calorimetric transition with apparent T m > 100°C was observed at basic pH (9.0 and 10.0). Urea-induced unfolding at pH 4.0, and at pH 2.0 with GuHCl, in presence of 8 M urea also reveals incomplete unfolding. Milin has been found to exhibit proteolytic resistant in either native or denatured state against various commercial proteases. These results imply that the high conformational stability of milin against various denaturating conditions enable its potential use in protease-based industries.
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Acknowledgments
The financial assistance to S.C.Y. from the Council of Scientific and Industrial Research, Government of India, in the form of a research fellowship and the financial assistance from UGC and DBT, Government of India, are acknowledged. Thanks are due to Prof. Rajiv Bhat, JNU for helping with DSC experiments and Prof. Wolfram Saenger, Institute of Crystallography, FU Berlin Germany for proteolytic experiments.
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Yadav, S.C., Jagannadham, M.V. Complete conformational stability of kinetically stable dimeric serine protease milin against pH, temperature, urea, and proteolysis. Eur Biophys J 38, 981–991 (2009). https://doi.org/10.1007/s00249-009-0490-5
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DOI: https://doi.org/10.1007/s00249-009-0490-5